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The  MICROSCOPE 

AN    INTRODUCTION  TO  MICROSCOPIC 
METHODS  AND  TO  HISTOLOGY 

BY  SIMON  HENRY  GAGE 

PROFESSOR   OF    HISTOLOGY    AND    EMBRYOLOGY,    EMERITUS 
IN  CORNELL    UNIVERSITY 


Convex 

Lens 

MAGNIFIER 

Microscope 

Objective 

Ocular 


THIRTEENTH  EDITION 
Revised  and  Illustrated  by  over  250  Text  Figures 

ITHACA,  NEW  YORK 
THE   COMSTOCK   PUBLISHING   COMPANY 

1920 


Copyright,  1908 

BY  SIMON  HENRY  GAGE 

All  Rights  Reserved 


Copyright,  1917 

BY  SIMON  HENRY  GAGE 

All  Rights  Reserved 

Copyright,  1920 

BY  SIMON  HENRY  GAGE 

All  Rights  Reserved 


TO 

SUSANNA  PHELPS  GAGE 

WHO  AS  A  STUDENT  RECEIVED  HELP  AND  INSPIRATION  FROM  THE  EARLIEST 
EDITIONS  OF  THIS  WORK,  AND  WHO  AIDED  IN  EVERY  WAY  POSSIBLE  BY 
DRAWING,  INDEX-MAKING  AND  LITERARY  CRITICISM  IN  IMPROVING  THE  LATER 

EDITIONS;  AND  WHO  NOW  HAS  ENTERED  INTO  THE  POSSIBILITIES  OF  ETERNITY 
THIS  BOOK  IS  DEDICATED 


17 


..iii  UK  i 


PREFACE 

IN  revising  and  rewriting  this  book  now  for  the  twelfth  time,  the  aim  has  been 
as  for  all  previous  editions,  to  give  the  student  the  benefit  of  the  fundamental 
things  which  have  been  worked  out  in  microscopy.  The  opportunities  given  by 
the  freedom  from  teaching  have  rendered  it  possible  to  make  this  revision  more 
thorough  than  could  be  done  in  any  previous  edition. 

Progress  in  all  that  pertains  to  microscopy  has  been  marked  during  the  last 
ten  years.  Any  one  can  see  this  clearly  by  comparing  the  catalogues  of  manu- 
facturers sent  out  ten  years  ago  with  those  sent  out  at  the  present  time. 

Nothing  fundamentally  new  has  appeared,  but  there  have  been  great  advances 
in  making  practical  and  usable  many  processes  and  much  apparatus  for  which 
the  basic  knowledge  has  existed  for  a  considerable  time.  Of  course  there  are  some, 
principles  and  manipulations  which  a  person  must  become  master  of  if  he  is  to  work 
successfully  with  the  microscope.  These  have  been  treated  mainly  as  in  the  past. 
Of  the  new  things  nothing  has  been  considered  in  the  book  which  has  not  been 
personally  tested  and  found  to  be  workable  and  helpful. 

Among  the  most  important  means  recently  made  available,  especially  for  students 
of  biology,  are  the  following: 

(1)  The  single  objective  binocular  for  all  powers  of  the  microscope  from  the 
lowest  to  the  highest. 

(2)  The  dark-field  illuminator  for  all  powers,  especially  the  highest  powers 
with  which  the  finest  details  in  living  structures  can  be  seen  with  marvelous  clear- 
ness.   This  makes  it  possible  to  compare  the  living  cell  with  the  fixed  and  stained 
one. 

(3)  The  perfection  of  apparatus  with  which  the  powerful  electric  lights  recently 
produced  have  become  available  for  demonstrations  and  for  drawing  with  the 
projection  microscope. 

(4)  The  perfection  of  photographic  light  filters  and  the  production  of  dry  plates 
sensitive  to  the  whole  spectrum  makes  it  possible  to  get  good  photographs  of  any 
microscopic  specimen,  and  indeed  of  any  specimen. 

(5)  From  the  numbers  who  are  affected,  and  the  extent  of  its  application,  per- 
haps the  greatest  improvement  of  all  has  been  the  production  of  a  glass  filter  which, 
when  used  with  a  gas  filled  mazda  lamp,  gives  a  light  of  true  daylight  quality  and 
of  sufficient  intensity  for  all  powers  of  the  microscope. 

In  preparing  this  edition  some  parts  of  the  previous  edition  have  been  omitted. 
For  example,  the  pages  on  micro-chemistry  and  metallography  have  been  left 


vi  PREFACE 

out  because  the  Micro- Chemistry  of  Dr.  E.  M.  Chamot,  which  has  recently  appeared, 
treats  these  and  indeed  all  matters  pertaining  to  the  chemico-physical  side  of 
microscopy  in  an  adequate  manner. 

As  a  closing  word  it  may  be  said  that  even  an  elementary  book  like  this  depends 
for  its  production  upon  many  helps.  The  work  of  others  must  be  looked  for  in  a 
great  library;  special  knowledge  in  allied  departments  must  be  utilized  through 
the  help  of  colleagues;  apparatus  and  ideas  can  only  be  put  in  graphic  form  by  the 
deft  hand  of  the  artist;  and  perhaps  most  important  of  all  is  the  advice  and  criti- 
cism of  the  friend.  All  of  these  helps  the  author  has  had  in  abundance,  and  he 
feels  grateful  to  each  helper. 

PREFACE  TO  THE  THIRTEENTH  EDITION 

BESIDES  some  verbal  and  numerical  corrections  in  this  edition,  added  emphasis 
has  been  placed  upon  Dark-field  Microscopy;  and  some  new  apparatus,  com- 
bined with  daylight  glass,  has  been  introduced  to  make  its  use  easier  and  more 
comfortable  for  the  eyes.  It  is  believed  that  this  powerful  agent  of  investigation 
deserves  very  wide  application.  The  advantages  of  a  dark  field  for  increasing 
visibility  has  been  appreciated  in  astronomy,  time  out  of  mind,  and  ever  since  the 
invention  of  the  microscope  (1590);  and  especially  for  the  modern  compound  mi- 
croscope since  the  introduction  of  Wenham's  paraboloid  reflector  (1850),  and  trun- 
cated paraboloid  (1856). 

The  Corning  Daylight  Glass  for  microscope  lamps  has  proved  such  a  boon,  that 
an  improved  lamp-house,  especially  designed  for  the  student  laboratory,  has  been 
worked  out  and  in  actual  use  during  the  last  college  year.  It  serves  not  only  for 
lighting  the  microscope,  but  for  note- taking,  drawing  and  reading  (figs.  37,  125) 
The  author  is  much  indebted  to  Dr.  B.  F.  Kingsbury  for  aid  in  perfecting  this  lamp. 

Attention  is  again  called  to  the  method  of  producing  line  drawings  from  photo- 
graphs (§§  289,  3i6a). 

It  is  hoped  that  the  discussion  of  the  units  of  measure  for  microscopic  objects 
and  for  light  waves  (§§  246,  406)  may  help  to  correct  the  confusion  in  this  field. 
Acknowledgement  is  gratefully  made  for  help  in  this  matter  by  the  U.  S.  Bureau 
of  Standards,  the  Cornell  University  Department  of  Physics,  and  the  Optical  De- 
partment of  the  Corning  Glass  Works. 

Finally,  for  progress  in  the  microscopical  field  attention  is  especially  called  to  the 
Symposium  on  the  Microscope;  its  design,  construction  and  applications,  held 
by  the  Royal  Microscopical  Society,  the  Faraday  Society,  the  Optical  Society  and 
the  Photographic  Society  in  cooperation  with  the  Technical  Optics  Committee  of 
the  British  Science  Guild,  in  the  rooms  of  the  Royal  Society,  Jan.  14,  1920.  The 
papers  and  discussions  at  the  Symposium,  and  the  discussions  since  held  in  Sheffield 
and  Glasgow,  and  also  a  report  of  the  discussion  held  in  the  Hall  of  the  Royal 
Microscopical  Society  in  April.  1920,  are  soon  to  be  published. 

CORNELL  UNIVERSITY  SIMON  HENRY  GAGE 

July  15,  1920 


CONTENTS 

PAGES       FIG. 

Introduction 1-5    1-3 

CHAPTER   I 6-35     4-29 

The  Microscope  and  Its  Parts:  Simple  and  compound  micro- 
scopes; Visual  angle  and  apparent  size  of  objects;  Lenses;  Con- 
struction of  images;  Experiments  with  the  simple  microscope; 
Compound  microscope  and  objectives  and  oculars;  Experiments 
with  the  compound  microscope. 


CHAPTER   II 36-76    3o-5of 

Practical  Work  with  the  Parts  of  the  Microscope:  Focusing 
experiments;  Working  distance;  Lighting  with  daylight;  Light- 
ing with  artificial  light;  Artificial  daylight  with  daylight  glass; 
Condensers  or  illuminators,  and  experiments  with  them;  Dark- 
ground  illumination  with  low  and  with  high  powers. 


CHAPTER   III   77-106     51-64 

Adjustable  and  Immersion  Objectives;  Binocular  Micro- 
scopes; Care  of  the  Microscope  and  of  the  Eyes:  Adjustable  and 
immersion  objectives  and  experiments;  Refraction  and  color 
images;  Binocular  microscopes  for  low  and  for  high  powers;  Care 
of  the  microscope  and  of  the  eyes;  Testing  the  microscope; 
Laboratory  and  school  microscopes;  Markers  and  mechanical 
stages;  Standard  sizes  of  the  Royal  Microscopical  Society. 


CHAPTER    IV    107-126     65-74 

Interpretation  of  Appearances  Under  the  Microscope:  Deter- 
mination whether  an  object  is  under  the  microscope  or  the 
appearance  is  due  to  dust,  etc.,  on  the  ocular,  etc.;  Relative 
position  of  objects;  Air  bubbles  and  oil  globules;  Velocity  under 
the  microscope;  Pedesis  or  Brownian  movement;  Inversion  of 
the  microscopic  image. 


viii  CONTENTS 


PAGES  FIG. 

CHAPTER   V 127-159     75-98 

Magnification  and  Micrometry  with  the  Microscope :  Need  of 
magnification;  Visual  angle  and  the  microscope;  Magnification 
and  virtual  and  real  images;  Distance  at  which  to  determine 
magnification;  Ways  to  determine  magnification;  Camera 
lucidae,  ocular  micrometers. 


CHAPTER   VI    160-205     99-125 

Drawing  and  Class  Demonstrations  with  the  Microscope  and 
Projection  Microscope:  Drawing  microscopic  objects  with  the 
camera  lucida  or  with  the  projection  microscope;  Determining 
the  magnification  at  which  the  drawing  is  made;  Avoidance  of 
inversion;  Use  of  photographic  prints  to  aid  in  making  draw- 
ings; drawings  and  their  lettering  for  publication;  Class  demon- 
strations. 

CHAPTER    VII 206-245     126-143 

Photography  with  the  Microscope  and  with  Projection  Appa- 
ratus: Photographs  of  embryos,  etc.,  with  a  vertical  camera; 
Photographing  with  a  microscope;  Lighting  and  experiments  in 
photography;  Scale  at  which  photographs  are  made;  Daylight 
glass  for  photographing  with  the  microscope;  Enlarging  photo- 
graphs; Color  screens  and  color  photography. 


CHAPTER    VIII    246-269     144-150 

The  Spectroscope  and  Polariscope  and  Then-  Use  with  the 
Microscope:  Visible  and  invisible  radiation;  Kinds  of  spectra; 
Micro-spectroscope  and  its  use;  Absorption  bands;  Micro- 
polariscope  and  its  use. 

CHAPTER   IX 270-311     151-184 

Some  Optical  Principles  Involved  hi  the  Construction  and  Use 
of  the  Microscope:  Reflection,  refraction,  dispersion,  diffraction; 
Lenses  and  images;  Spherical  and  chromatic  aberration  and  their 
.correction;  Angular  and  numerical  aperture;  Diffracted  light 
in  microscopy;  Magnification  of  the  individual  parts  of  the 
microscope;  Parfocal  oculars  and  objectives. 


CONTENTS  ix 

PAGES  FIG. 

CHAPTER   X 312-367     185-215 

Materials  of  Microscopy;  Mounting  and  Storing  Microscopic 
Specimens:  Slides  and  cover-glasses,  their  size  and  preparation; 
Mounting  microscopic  objects  in  the  various  ways;  Isolation  of 
tissue  elements;  Collection  and  study  of  microscopic  animals  and 
plants;  Labeling,  cataloguing  and  storing  microscopic  prepara- 
tions; Cabinets,  lockers  and  trays  for  specimens;  Reagents  for 
microscopic  work,  with  formulae  for  their  preparation. 

CHAPTER   XI 368-423     216-247 

Fixing,  Imbedding,  Sectioning,  Staining;  Serial  Sections  and 
Models:  Fixation  and  preservation  of  organs,  tissues,  embryos 
and  small  animals  for  microscopic  study;  Microtomes  and  sec- 
tion knives  for  microscopic  sections;  Free-hand  sectioning. 
Paraffin  method  of  sectioning;  Collodion  method  of  sectioning; 
Staining  and  mounting;  Serial  sectioning  and  its  advantages; 
Serial  sections  of  embryos  and  small  animals;  Orientation  for 
sectioning  and  mounting  the  series;  Planes  for  sectioning,  — 
transverse,  saggital  and  frontal;  Modeling  with  wax,  and  with 
blotting  paper.  Drawings  for  models. 

CHAPTER   XII 424-443     248-252 

Brief  History  of  Lenses  and  Microscopes:  Lenses  and  their 
use  for  spectacles,  for  projection  apparatus;  Simple  microscopes; 
Dutch  compound  microscope;  Keplerian  compound  microscope; 
Binocular  microscopes ;  Microscopes  where  two  or  more  ob- 
servers can  look  at  the  same  object  at  the  same  time;  Mirrors 
and  condensers;  Achromatization;  Immersioji  and  homoge- 
neous immersion  objectives;  Projection  microscope;  Drawing 
magnified  images  by  the  aid  of  the  camera  lucida  and  projection 
apparatus. 

Bibliography 445~452 

Index 453-472 


THE    MICROSCOPE 
AND    MICROSCOPICAL    METHODS 

INTRODUCTION 

IN  dealing  with  the  possibilities  and  use  of  any  method  of  investi- 
gation, any  machine  or  piece  of  scientific  apparatus,  the  writer  or 
teacher  will  naturally  proceed  as  seems  to  him  best  from  his  personal 
experience,  from  his  general  theory  of  education,  and  from  his  con- 
ception of  the  style  and  method  of  presentation  which  will  render 
his  book  most  acceptable  to  his  possible  readers. 

As  stated  in  the  preface  to  the  sixth  edition,  this  book  had  its  origin 
in  the  laboratory,  and  its  purpose  was,  and  still  is,  to  give  the  guidance 
by  which  those  unfamiliar  with  the  microscope  and  the  methods  of 
work  with  it  can  gain  an  intelligent  understanding  of  the  instrument, 
its  limitations,  and  its  possibilities  for  aiding  one  to  arrive  at  truth. 

In  working  out  the  plan  the  following  landmarks  have  been  kept 
constantly  in  sight: 

(i)  To  most  minds,  and  certainly  to  those  having  any  grade  of 
originality,  there  is  a  great  satisfaction  in  understanding  principles; 
and  it  is  only  when  the  principles  are  firmly  grasped  that  there  is 
complete  mastery  of  instruments,  and  full  certainty  and  facility  in 
using  them.  The  same  is  true  of  the  methods  of  preparing  objects 
for  microscopic  study,  and  the  interpretation  of  their  appearances 
when  seen  under  the  microscope. 

Much  good  work  can  be  and  has  been  done  by  the  rule  of  thumb 
method,  in  which  there  is  no  real  understanding  of  the  underlying 
reason  for  any  of  the  operations;  the  worker  simply  knows  that  good 
results  follow  a  certain  course  of  action.  Probably  most  of  the  work 
of  the  world  is  done  by  rule  of  thumb.  But  the  originators  of  the 
knowledge  making  rule  of  thumb  possible  must  have  some  compre- 


INTRODUCTION 


[INTRO, 


hension  of  principles,  and  the  reasons  for  what  is  done.     For  creative 
work,  then,  knowledge  of  principles  is  indispensable. 

(2)  Need  of  abundant  practical  work  to  go  with  the  theoretical 
part  has  been  shown  by  all  human  experience.  In  all  the  crafts  and 
in  all  the  fine  arts  mastery  comes  only  with  almost  endless  effort 
and  repetition,  the  most  common  example  being  the  attainment  of 
facility  in  music.  Hence  in  this  work  there  have  been  introduced 
many  practical  exercises  so  that  the  worker  might  gain  the  deftness 
needed.  It  is  also  a  part  of  human  experience  that  in  successfully 
going  through  the  manipulations  necessary  to  demonstrate  principles 


FIG.  i. 


PROJECTION  MICROSCOPE  WITH  ENLARGED  REAL  IMAGE 
ON  THE  SCREEN. 


and  methods,  the  principles  and  methods  themselves  become  more 
real.  That  is,  comprehension  of  principles  aids  in  the  certainty  with 
which  work  can  be  done,  and  conversely  the  doing  of  the  work  helps 
to  increase  the  grasp  on  the  principles. 

After  observing  the  work  of  students  in  my  own.  and  in  other  labora- 
tories the  conclusion  was  reached  and  expressed  in  the  third  edition 
of  this  book  (1891)  that  "simply  reading  a  work  on  the  microscope, 
and  looking  a  few  times  into  an  instrument  completely  adjusted  by 
another,  is  of  very  little  value  in  giving  real  knowledge.  In  order 
that  the  knowledge  shall  be  made  alive,  it  must  be  a  part  of  the 
-student's  experience  by  actual  experiments  carried  out  by  the 
student  himself." 

Beale,  in  his  work  on  the  microscope,  expresses  it  thus:  "  The  num- 


INTRO.] 


INTRODUCTION 


her  of  original  workers  emanating  from  our  schools  will  vary  as  prac- 
tical work  is  favored  or  discouraged.  It  is  certain  that  they  who  are 
most  fully  conversant  with  elementary  details, 
and  most  clever  at  demonstration,  will  be  most 
successful  in  the  consideration  of  the  higher  and 
more  abstruse  problems,  and  will  feel  a  real  love 
for  their  work  which  no  mere  superficial  inquirer 
will  experience.  It  is  only  by  being  thoroughly 
grounded  in  first  principles,  and  well  practised 
in  mechanical  operations,  that  any  one  can  hope 
to  achieve  real  success  in  the  higher  branches  of 
scientific  inquiry,  or  to  detect  the  fallacy  of 
certain  so-called  experiments." 

And  Hon.  J.  D.  Cox,  skilled  alike  in  the  arts  of 
war,  statesmanship,  and  science,  in  his  notable 
address  upon  Systematic  Instruction  in  the  Micro- 
scope at  the  University,  before  the  American 
Microscopical  Society,  in  1893,  says:  "I  wish  to 
urge  the  desirability  of  a  somewhat  extensive 
course  of  technical  training  in  regard  to  the  micro- 
scope. .  .  .  Any  one  who  desires  to  devote  him- 
self seriously  to  investigation  with  the  microscope 
will  find  great  advantage,  as  it  seems  to  me,  in  de- 
voting some  time  to  the  study  of  the  instrument 
itself  in  all  its  parts,  and  the  history  of  their  de- 
velopment." The  study  of  this  whole  address  is 
urged  upon  the  person  interested  in  the  just  ap- 
preciation of  the  different  parts  of  the  microscope 
and  their  successful  employment  or  improvement. 

Sir  A.  E.  Wright,  in  his  book  "Principles  of 
Microscopy,"  says  this:  "Every  one  who  has  to 
use  the  microscope  must  decide  for  himself  the 
question  as  to  whether  he  will  do  so  in  accordance 
with  a  system  of  rule  of  thumb,  or  whether  he 
will  seek  to  supersede  this  by  a  system  of  reasoned  action  based  upon 
a  study  of  his  instrument  and  a  consideration  of  the  scientific  prin- 


Object 


FIG.  2.  A  SIMPLE 
MICROSCOPE  HELP- 
ING THE  EYE  TO 
FORM  A  RETINAL 
IMAGE  OF  A  NEAR 
OBJECT. 

Object  The  object 
to  be  seen  by  the 
eye. 

Lens  The  double 
convex  lens  acting 
as  a  magnifier  or 
simple  microscope 
to  aid  the  eye  in  see- 
ing a  near  object. 

Cornea  The  cor- 
nea of  the  eye. 

r  The  single  re- 
fracting surface  in 
the  schematic  eve. 

cl  The  crystalline 
lens  of  the  eye,  also 
the  center  of  the  re- 
fracting surfaces  or 
the  nodal  point  of 
the  eye  where  the 
secondary  axial  rays 
cross. 

ri  Retinal  image; 
it  is  inverted. 


INTRODUCTION 


[INTRO. 


rs 


ciples  of  microscopical  technique.     The  present  text-book  [his  "Prin- 
ciples of  Microscopy'']  has  no  message  to  those  who  are  content  to 
follow  a  system  of  rule  of  thumb,  and  to  eke  this  out  by  blind  trial 
and  error.     It  addresses  itself  to  those  who 
are  dissatisfied  with  the  results  thus  obtained 
and  who  desire  to  master  the  scientific  prin- 
ciples of  microscopy,  even  at  the  price  of 
some  intellectual  effort." 

From  the  observation  of  ten  generations 
of  students  and  their  subsequent  career  I  am 
confirmed  in  the  belief  that  for  attainment 
in  study  with  the  microscope,  as  in  all  other 
human  endeavor,  a  person  must  pay  for  all 
he  gets. 

(3)  In  considering  the  microscope,  it  may 
be  looked  at  as  a  machine  composed  of  glass 
and  brass  complete  in  itself,  or  it  may  be 
considered  as  an  artificial  aid  to  the  eye, 
like  a  spectacle.  When  complete  in  itself 
it  is  properly  called  a  projection  microscope, 


OBJECTIVE 


r-.f 

Object 


MIRROR 


FIG.  3.  A  COMPOUND  MICROSCOPE  HELPING 
THE  EYE  TO  FORM  A  RETINAL  IMAGE  OF  A  NEAR 
OBJECT. 

Mirror  The  plane  and  concave  mirror  to  re- 
flect light  through  the  object. 

Object    The  small  object  to  be  seen  by  the  eye. 

Objective  The  objective  of  the  compound  micro- 
scope to  form  a  real  image  of  the  small  object. 

Axis  The  principal  optic  axis  of  the  micro- 
scope. 

/  Principal  focus  of  the  ocular  and  of  the  ob- 
jective. 

r  im     The  real  image  formed  by  the  objective. 

Ocular  The  double  convex  lens  enabling  the 
eye  to  see  the  real  image  formed  by  the  objective. 

cr     The  cornea  of  the  eye. 

rs    The  refracting  surface  of  the  schematic  eye. 

L     The  crystalline  lens  of  the  eye. 

r  i  The  retinal  image;  it  is  erect  with  reference 
to  the  object,  but  inverted  as  compared  with  the 
real  image. 


INTRO.]  INTRODUCTION  5 

for  it  produces  an  image  wholly  independent  of  the  eye  of  tbe  ob- 
server. This  image  may  be  fixed  on  a  photographic  plate  or  used  as 
a  basis  for  a  drawing  (fig.  i).  On  the  other  hand,  when  used  as  a 
microscope  in  the  ordinary  way,  the  eye  of  the  observer  is  an  integral 
part  of  the  optical  combination,  just  as  integral  a  part  as  the  objec- 
tive or  the  ocular  (fig.  2,  3).  This  being  the  case  the  optical  perfec- 
tion of  the  eye  is  as  influencing  on  the  final  retinal  image  as  the 
perfection  of  the  other  optical  parts. 

And  finally,  quoting  again  from  the  preface  of  the  third  edition, 
"In  considering  the  real  greatness  of  the  microscope  and  the  truly 
splendid  service  it  has  rendered,  the  fact  has  not  been  lost  sight  of 
that  the  microscope  is,  after  all,  only  an  aid  to  the  eye  of  the  observer, 
only  a  means  of  getting  a  larger  image  on  the  retina  than  would  be 
possible  without  it,  but  the  appreciation  of  this  retinal  image,  whether 
it  is  made  with  or  without  the  aid  of  a  microscope,  must  always 
depend  upon  the  character  and  training  of  the  seeing  and  appreciating 
brain  behind  the  eye.  The  microscope  simply  aids  the  eye  in  furnish- 
ing raw  material,  so  to  speak,  for  the  brain  to  work  upon." 


CHAPTER  I 
THE  MICROSCOPE  AND  ITS  PARTS 

§  1.    Apparatus  and  material  for  Chapter  I. 

1.  A    simple  microscope   (§  3,   14,  6.  Stage    micrometer    (§  48). 

fig.  4,  6).  7.  Homogeneous  immersion  liquid 

2.  A    compound    microscope    with       (§  25). 

nose-piece  (§4,  fig.   25-28).  8.  Mounted  letters  or  figures  (§  50). 

3.  Eye-shade  (fig.  33).  9.  Ground-glass    and    lens    paper 

4.  Achromatic      (§  26),     apochro-       (§  50). 

matic  (§  28),  dry  (§  22),  immersion  10.  Black  card  with  pin-hole  (§  7, 

(§  23-25),    unadjustable    (§  30),   ad-       fig.  7). 

justable  objectives  (§  31).  n.  Dissecting    spectacles    (§  145). 

5.  Huygenian  or  negative   (§  38), 
positive    (§39),    compensation    ocu- 
lars (§40). 

§  2.  As  the  word  itself  indicates,  a  microscope  is  an  instrument 
with  which  one  can  see  small  things  (§  2a). 

The  microscope  makes  small  things  or  minute  details  of  larger 
things  visible  in  two  distinct  ways,  both  ways  being  dependent  on  an 
increase  of  the  visual  angle  (§  6,  226-227,  fig.  75-76). 

(1)  The  first  way  of  increasing  the  visual  angle  and  thus  making 
small  things  or  details  visible  is  by  means  of  one  or  more  lenses  used 
as  a  kind  of  spectacle  by  which  the  eye  is  enabled  to  form  a  sharp 
image  on  the  retina  when  optically  so  close  to  the  object  that  with- 
out the  artificial  aid  a  sharp  image  on  the  retina  could  not  be  pro- 
duced (fig.  2,  3,  6). 

(2)  The  second  way  of  increasing  the  visual  angle  under  which 
small  things  or  details  are  viewed  is  by  means  of  a  projection  micro- 
scope, which,  wholly  independent  of  the  eye,  produces  a  sharp,  greatly 
enlarged  image  of  the  object  upon  a  white  surface.    The  eye  then 
looks  at  this  image  as  though  it  were  the  object  itself  and  of  that  size 
(fig.  i,  §312). 

The  fundamental  difference  in  the  two  forms  of  microscope  is  that 

6 


CH.  I] 


SIMPLE  AND   COMPOUND  MICROSCOPES 


in  the  first  only  a  retinal  image  is  formed,  while  in  the  second,  a  screen 
image  and  from  that  a  retinal  image. 

In  this  book  the  first  form  of  microscope  is  mainly  considered  except 
in  Ch.  VI  and  VII,  where  the  projection  microscope  is  much  used. 


Simple    Microscope 


§  2a.  The  word  Microscope  is 
from  two  Greek  words:  /"K/>6s — 
mikros,  small,  and  ffKoireiv  — 
skopein,  to  see.  The  word  was 
compounded  and  given  a  Latin 
form  by  Giovanni  Faber  of  the 
Academy  of  the  Lincei,  as  shown 
by  a  letter  of  his  to  Cesi,  Presi- 
dent of  the  Lyceum,  dated  April 
13, 1625.  Faber  says:  "  As  I  also 
mention  his  [Galileo's]  new 
occhiale  to  look  at  small  things 
and  call  it Microscopium.  "Jour. 
Royal  Microscopical  Society, 
1889,  p.  578;  Carpenter-Dallin- 
ger,  p.  125. 

SIMPLE  AND  COMPOUND 
MICROSCOPES 

§  3.  A  simple  microscope 
or  magnifier  is  a  lens  or  a 
combination  of  lenses  to  use 
with  the  eye,  and  with  it  an 
enlarged,  erect  image  is  seen, 
that  is,  the  enlarged  image 
has  all  its  parts  in  the  same 
position  as  in  the  object  it- 
self (fig.  4),  and  but  one 
image  is  formed,  and  that  is  formed  upon  the  retina. 

§  4.  A  compound  microscope  is  one  in  which  a  lens,  or  combination 
of  lenses,  called  an  objective,  forms  a  real  image,  and  this  real  image 
is  looked  at,  as  if  it  were  an  object,  by  the  eye  and  a  magnifier  or 
simple  microscope  known  as  an  ocular.  The  image  seen  has  the 
object  and  its  parts  inverted.  In  the  compound  microscope  two 
images  are  formed,  one  by  the  objective  independent  of  the  eye,  and 
the  other  on  the  retina  by  the  action  of  the  eye-lens  of  the  ocular  and 
the  cornea  and  crystalline  lens  of  the  eye  (fig.  3). 


Convex 

Lens 

MAGNIFIER 
Microscope 
Objective 
Ocular 


FIG. 


4.     FINE  PRINT  SEEN  BY  THE  UNAIDED 
EYE  AND  THROUGH  A  MAGNIFIER. 


8 


SIMPLE  AND   COMPOUND  MICROSCOPES 


[Cn.I 


§  5.    Virtual   images.  —  In   all   diagrammatic   drawings   showing 
the  microscope  when  looking  directly  into  it,  an  enlarged,  imaginary 


FIG.  5-6. 


Object   >.^^ 

VISION   BY  THE  UNAIDED   EYE  AND  BY  THE   AID   OF   A 
MICROSCOPE. 


SIMPLE 


FIG.  5.     UNAIDED  EYE  VISION.   Axis,  THE  PRINCIPAL  OPTIC  Axis  OF  THE 
EYE  EXTENDED  TO  THE  OBJECT. 

Object  The  object  to  be  seen;  it  is  at  a  distance  of  250  millimeters  from  the 
eye. 

r  i     The  retinal  image;    it  is  inverted. 

FIG.  6.     VISION  BY  THE  AID  OF  A  SIMPLE  MICROSCOPE.     Axis,  PRINCIPAL 
OPTIC  Axis  OF  THE  MICROSCOPE  AND  OF  THE  EYE. 

A1  Bl     The  object  within  the  principal -focus  (F)  of  the  lens. 

S  M     A  double  convex  lens  acting  as  a  simple  microscope. 

Cr     The  cornea  of  the  eye. 

R     Single  refracting  surface  of  the  schematic  eye. 

L     The  crystalline  lens  of  the  eye. 

Bz  A2     The  retinal  image;    it  is  inverted. 

A3  B3  The  virtual  image  projected  into  the  field  of  vision  at  250  milli- 
meters; it  is  erect,  and  the  appearance  is  exactly  as  if  the  virtual  image  were 
an  object  as  in  fig.  4,  and  no  lens  were  present. 

object  is  shown  out  in  space.  This  is  frequently  called  a  virtual  image. 
If  there  were  no  microscope  and  an  object  of  that  size  were  in  front 
of  the  observer,  he  would  get  the  same  appearance,  for  a  retinal  image 
of  the  same  size  would  be  produced  as  is  produced  by  the  magnifying 


CH.  I]  VISUAL  ANGLE  9 

glass  helping  the  eye  (fig.  5-6).  In  the  projection  microscope  there 
is  an  actual  or  real  enlarged  image  on  a  screen  which  the  observer 
looks  at  as  if  it  were  a  large  object  (fig.  i).  If  one  keeps  in  mind  that 
virtual  images  are  purely  imaginary,  and  that  real  images  are  pro- 
duced by  actual  rays  of  light,  it  will  help  to  avoid  confusion  and 
wrong  interpretations.  In  every  case  where  an  object  is  seen,  light 
rays  must  pass  from  the  object  to  the  eye,  and  these  rays  entering 
the  eye  must  form  an  image  on  the  retina.  It  is  the  retinal  image 
which  furnishes  the  brain  the  stimulus  for  vision. 

.  APPARENT  SIZE  OF  OBJECTS 

Whether  one  is  using  a  microscope  or  not,  the  apparent  size  of  any 
object  seen  depends  upon  the  visual  angle.  Practically  the  entire 
purpose  subserved  by  the  microscope  is  that  it  enables  the  eye  to  see 
objects  under  a  greater  visual  angle  than  would  be  possible  without 
the  artificial  aid. 

§  6.  Visual  angle.  —  This  is  the  angle  made  by  the  border  rays 
of  light  from  the  object  to  the  retina,  and  crossing  at  the  nodal  point 
or  optical  center  of  the  eye  (fig.  75-76). 

As  the  visual  angle  depends  upon  the  distance  the  object  is  sepa- 
rated from  the  eye,  any  means  by  which  the  object  can  be  brought 
closer  to  the  eye  will  result  in  giving  a  larger  apparent  size  to  the 
object,  or  in  magnifying  it.  The  lenses  of  the  microscope  used  with 
the  eye  enable  it  to  get  very  close  to  the  object  and  thus  increase  the 
visual  angle,  and  depending  on  the  closeness,  finer  and  finer  details 
of  the  object  are  separated,  for  they  subtend  an  angle  of  one  minute 
or  more  (see  §  226),  and  the  object  as  a  whole  has  a  much  greater 
apparent  size.  (For  further  discussion  see  Ch.  V.) 

§  7.  Pin-hole  card.  —  Use  a  piece  of  paper  about  the  size  of  a 
library  card.  If  the  slip  is  black  or  of  a  dark  color  it  makes  the  experi- 
ment a  little  easier  than  when  white  paper  is  used.  Make  a  hole  in 
this  with  a  needle  (fig.  7).  If  now  one  holds  the  slip  up  close  to  the 
eye  and  gets  the  hole  in  .the  optic  axis,  the  eye  can  see  brilliantly 
lighted  objects  very  clearly.  If,  to  start  with,  the  object  is  off  about 
i  meter,  quite  an  extent  of  it  can  be  seen,  and  it  will  appear  small. 
Now  go  up  closer  and  closer,  and  still  the  object  is  clearly  seen,  and 


10 


VISUAL  ANGLE 


[CH.I 


constantly  appearing  larger.  The  closer  one  gets  the  smaller  is  the 
visible  field,  but  the  larger  will  the  parts  seem  to  be.  If  the  hole  is 
quite  small,  one  can  get  the  object  within  4  or  5  cm.  of  the  eye  and  still 
see  the  image  clearly,  and  see  details  which  could  not  be  seen  at  a 
greater  distance. 

As  shown  in  the  figures  of  the  visual  angle  (fig.  76),  the  closer  the  eye 
gets  to  the  object  the  greater  will  be  the  visual  angle,  hemce  details 

are  shown  which  did  not  appear  at  a 
greater  distance.  One  of  the  best  meth- 
ods of  trying  this  experiment  is  to  use  for 
object  a  small  mark  made  wifrh  ink  or  a 
glass  pencil  on  a  window  or  on  a  milky 
or  transparent  lamp  shade.  Then  there 
will  be  plenty  of  light.  The  physiologi- 
cal explanation  of  the  power  to  see 
clearly  through  the  pinhole  at  a  distance 
of  5  cm.,  when,  if  the  eye  looks  directly 
at  the  object,  it  should  be  about  25  cm. 
from  the  eye,  is,  that  with  the  pin-hole 
the  beam  is  so  narrow  that  it  affects  So 
narrow  a  circle  on  the  retina  that  the 
appearance  is  like  a  good  focus.  If  one 
takes  away  the  card,  the  beam  gets  very 
wide  and  the  eye  has  only  a  blurred 
impression,  the  diffusion  circles  are  so 
large. 

§  7a.  In  case  one  loses  his  spectacles  or  has  the  accommodation  paralyzed 
by  atropin  for  testing  the  eyes,  it  is  possible  to  read  fairly  well  with  the  per- 
forated card  if  the  print  is  in  a  brilliant  light.  The  field  which  can  be  seen  at 
one  time  is  very  small,  so  one  must  move  the  print  or  the  head  almost  constantly. 

LENSES 

The  usual  and  most  effective  means  for  increasing  the  visual  angle 
when  examining  small  objects  is  by  the  use  of  lenses,  singly  or  in  com- 
bination. 

§  8.  Lens.  —  A  lens  means  a  mass  of  transparent  glass  or  other 
substance  with  one  plane  and  one  curved,  or  with  two  curved  sur- 


FIG.  7.     PIN-HOLE  CARD  FOR 
VIEWING  NEAR  OBJECTS. 


CH.  I] 


REFRACTION  AND  LENSES 


ii 


faces.     Natural  transparent  minerals  may  also  be  given  a  lenticular 
form,  e.g.  fluorite,  quartz,  etc. 

The  lens  is  usually  a  segment  of  a  sphere  or  of  two  spheres  (fig.  8). 
In  dealing  with  lenses  mention  must  frequently  be  made  of  the  optical 
center  of  the  lens,  the  principal  axis,  secondary  axis,  and  the  principal 
focus.  These  are  illustrated  in  fig.  8,  11-12,  and  are  briefly: 

(1)  Optical  center.  —  The  point  in  or 
near  a  lens  through  which,  if  rays  pass, 
they  will  suffer  no  angular  deviation,  and 
the  emerging  ray  will  be  parallel  to  the 
incident  ray  (fig.  8  c.l) . 

(2)  Principal  axis.  —  The  axis  passing 
through  the  centers  of  curvature  of  the 
two  spheres  whose  surfaces  bound  the 
lens  (fig.  8). 

(3)  Secondary  axis.  — Any  axis  oblique 
to  the  principal  axis,  but  passing  through 
the  optical  center  of  the  lens  (fig.  11-12). 
A  ray  along  a  secondary  axis  undergoes 
no  angular  deviation,  although  it  may 
suffer  displacement  as  a  ray  in  travers- 
ing a  piece  of  plane  glass  (fig.  51). 

(4)  Principal  focus.  —The  point  where 
rays  of  light,  parallel  to  the  principal  axis, 
cross  after  traversing  the  lens  (fig.  10). 
Every  lens  has  two  principal  foci,  one  on 
each  side  (fig.  10). 

With  concave  lenses  the  foci  are  virtual  (fig.  9). 

§  9.  Refraction.  —  By  this  is  meant  the  change  in  direction  of 
light  in  passing  from  one  transparent  medium  into  another.  The 
possibility  of  the  production  of  images  by  lenses  depends  upon  re- 
fraction. 

The  amount  of  refraction  depends  upon  two  things: 

(i)  The  difference  in  density  of  the  two  media.  The  greater 
the  difference,  the  greater  the  amount  of  bending  of  the  light  in 
passing  from  one  medium  to  another. 


FIG.  8.  LENS  WITH  OUT- 
LINES OF  THE  Two  SPHERES 
OF  WHICH  IT  is  SEGMENTS. 

Axis  The  principal  optic 
axis,  the  line  joining  the  two 
centers  of  curvature  (c  c'). 

c  c'  Centers  of  curvature, 
—  centers  of  the  two  spheres 
from  which  the  lens  is  de- 
rived. 

r  r'     Parallel  radii. 

/  /'  Tangents  at  the  term- 
inal points  of  the  radii. 

d  Center  of  the  lens,  — 
point  where  the  line  joining 
the  radii  at  the  tangential 
points  crosses  the  principal 
axis. 


12 


GEOMETRICAL  CONSTRUCTION  OF  IMAGES 


[CH.I 


FIG.  9.  CONCAVE  LENS 
SHOWING  VIRTUAL  Focus 
(F). 


(2)    The  obliquity  with  which  the  light  strikes  the  second  medium. 

The  greater  this  obliquity  the  greater  the  bending  of  the  light,  in 

accordance  with  the  law  of  sines.     (For 
further  discussion  see  Ch.  IX.) 

§  10.  Geometrical  construction  of  im- 
ages. —  In  this  book  the  lenses  shown  are 
thick,  but  the  course  of  the  rays,  for  sim- 
plicity, is  shown  to  be  as  if  the  lenses  were 
infinitely  thin,  that  is,  they  show  all  the 
bending  at  one  plane  (the  refracting  plane, 
fig.  11-12).  In  reality  there  is  one  refrac- 
tion at  the  incident  or  entering  surface  and 
one  at  the  emerging  surface.  With  thick 
lenses  like  those  figured,  there  will  be  no 

angular  deviation  for  rays  traversing  the  optical  center  of  the  lens, 

but  there  will  be  a  cer- 
tain   amount    of    dis-  „  \\\|/// 

placement,   although 

the  emerging   ray  will 

remain  parallel  to  the 

entering  or  incident  ray 

(fig.  si). 

For  the  construction 
of  images  it  is  necessary 
to  know  the  position  of 
the  principal  focus  and 
the  optical  center  of  the 
lens. 


FIG.  10. 


LENS  WITH  A  PRINCIPAL  Focus  ON 
EACH  SIDE. 


§  lOa.  Geometrical  con- 
struction of  images.  —  It 
should  be  remembered  in 
making  the  drawings  for 
the  geometrical  construc- 
tion of  images  that  there 
are  two  fundamental  laws 
which  must  always  be 
obeyed. 

(i)    Light  rays  extend  in  straight  lines  in  a  transparent  medium  of  uniform 
density,  and  whenever  the  direction  is  to  be  changed  the  light  must  meet  a 


Axis     The  principal  optic  axis. 

F  The  principal  focus,  —  the  point  on  the  axis 
at  which  rays  parallel  with  the  principal  axis 
cross. 

The  arrows  indicate  the  direction  of  the  light. 


CH.I] 


GEOMETRICAL  CONSTRUCTION  OF  IMAGES 


different  refracting  medium,  or  a  reflecting 
surface.  That  is,  the  direction  of  a  ray  of 
light  may  be  changed  by  using  a  mirror,  or 
by  putting  in  its  path  a  transparent  medium 
of  greater  or  less  refracting  power. 

(2)  The  second  law  is,  that  objects  are 
always  seen  in  the  direction  in  which  the 
light  reaches  the  eye,  regardless  of  the  act- 
ual position  of  the  object.  This  will  be 
abundantly  illustrated  in  the  chapter  on 
drawing;  and  every  one  knows  that  objects 
seen  in  a  mirror  are  not  where  they  appear 
to  be  in  the  mirror. 

§  11.     Construction  of  real  images. 
—  (i)   The  object  must  be  situated 
at  a  greater  or  less  distance  beyond 
the  principal  focal  point  (fig.  n). 

(2)  From  some  point  in  the  object, 
draw  a  line  to  the  refracting  plane  of 
the  lens  (§  10)  parallel  to  the  principal 
axis,  and  from  this  crossing  point  at 
the  refracting  plane  of  the  lens  to  the 
focus  of  the  lens,  and  continue  the  line 
indefinitely  (fig.  n). 

(3)  From  the  same  point  of  the  ob- 
ject as  in  (2),  draw  a  secondary  axis 
through  the  optical  center  of  the  lens 
and  extend  it  indefinitely  (fig.  n). 

The  image  of  the  point  in  the  object 
from  which  the  two  lines  were  drawn 
will  be  located  at  the  point  where  the 
two  extended  lines  cross  above  the  lens 
(fig.  n). 

The  image  of  all  the  other  points  of 
the  object  may  be  determined  by 
drawing  lines  from  them  exactly  as 
just  described. 

If  the  image  is  known  one  can  find 
the  object  by  reversing  the  process 
just  described. 


FIG.  ii-i2.  GEOMETRICAL  CON- 
STRUCTION OF  REAL  AND  OF  VIR- 
TUAL IMAGES. 

Object,  Object  The  object  of 
which  an  image  is  to  be  formed. 

Axis,  Axis  The  principal  optic 
axis  extended  above  and  below 
the  lens  to  the  object  and  image. 

S  Axis,  S  A xis  Secondary  axis 
passing  from  the  object  through 
the  center  of  the  lens. 

fy  />  />  /  Tne  principal  foci  of 
the  two  lenses. 

r-p  The  plane  of  refraction 
(the  ideal  plane  at  which  all  the 
refraction  is  made  to  occur  in 
diagrams  of  thick  lenses). 

R.  Image     Real  image. 

V.  Image  Virtual  image  indi- 
cated by  broken  lines  as  it  has  no 
real  existence. 

o  b,  r  m  Rays  of  light  indi- 
cated by  lines  passing  from  the 
extremities  of  the  object  to  the 
extremities  of  the  real  image, 
which  is  inverted. 

o  b,  i  2,  3  4,  v  m  Lines  rep- 
resenting rays  of  light  from  the 
object  passing  in  a  diverging 
manner  above  the  lens,  and  ex- 
tended by  broken  lines  below  the 
lens  to  form  a  virtual  image  at 
their  crossing  points,  v  m. 


GEOMETRICAL  CONSTRUCTION  OF  IMAGES 


[Cn.  I 


§  12.     Construction  of  virtual  images.  —  (i)   For  these  the  object 
must  be  somewhere  between  the  principal  focus  and  the  lens. 

(2)   From  some  point  in  the  object  draw  a  line  to  the  refracting 
plane  of  the  lens,  parallel  to  the  principal  axis,  and  from  this  point 

through  the  principal 
focus,  and  continue  it 
indefinitely. 

(3)  From  the  same 
point  of  the  object  as 
in  (2)  draw  a  secondary 
axis  through  the  optical 
center  of  the  lens  and 
extend  it  indefinitely. 

The  two  lines  will  not 
cross  above  the  lens,  but 
if  they  are  extended  be- 
low the  lens  (fig.  12) 
they  will  cross,  and  the 
crossing  point  locates 
the  image.  But  as  there 
are  no  light  rays  ex- 
tending in  this  direction 
the  image  is  imaginary 
or  virtual.  That  is,  it 


FIG.  13-14.     REAL  IMAGE  WITH  THE  OBJECT  FAR 
FROM  AND  NEAR  TO  THE  PRINCIPAL  FOCUS. 


Axis,  Axis     The  principal  optic  axis  extended 
above  and  below  the  lenses. 

/>  />  />  /     The  principal  foci  of  the  lenses. 

L  c,  L  c     The  same  lens  with  the  object  farther 
from  and  nearer  to  its  principal  focus. 

A  B,  B'  A'     The  object  and  its  inverted  image 
when  the  object  is  far  from  the  principal  focus. 

A  B,  B'  A '     The  object  and  larger  inverted  real 
image  when  the  object  is  near  the  principal  focus. 


looks  as  if  the  rays 
reaching  the  eye  orig- 
inated from  the  point 
where  the  rays  would 
cross  if  extended  back- 


ward. 

§  13.  Relative  position  of  object  and  image.  —  The  general  law 
is  that  the  nearer  the  object  to  the  principal  focus  the  farther  away 
is  the  image;  and  conversely,  the  nearer  the  image  is  to  the  principal 
focus  the  farther  from  it  must  be  the  object.  And  from  the  law  of 
similar  triangles,  the  size  of  the  image  is  to  the  size  of  the  object  as 
the  distance  of  the  image  from  the  center  of  the  lens  is  to  the  distance 


CH.  I] 


SIMPLE   MICROSCOPE   EXPERIMENTS 


of  the  object  from  that  center.  In  a  word,  the  nearer  the  object  to 
the  focus,  the  farther  away  the  image  from  that  point,  and  the  greater 
the  relative  size  of  the  image.  This  is  equally  true  of  real  and  of 
virtual  images  (fig.  13-16). 


EXPERIMENTS  WITH  THE  SIMPLE  MICROSCOPE 

§  14.    For  a  simple  microscope  use  a  reading  glass,  or  any  form  of 
simple  microscope  such  as  the  tripod  magnifier  (fig.  17,  18).     Hold 
the  magnifier  over  a  printed 
page  and   look   through   the 
magnifier.     The  letters  and 
words  will  appear  as  they  do 
with  the  naked  eye,  but  larger 
(fig.  4). 

In  order  to  get  the  sharpest 
image  it  will  be  necessary  to 
raise  and  lower  the  magnifier 
until  the  best  position  is 
found.  This  mutual  arrange- 
ment of  magnifier  and  object 
is  called  focusing,  or  getting 
into  focus. 

§  15.     Size  of  the  field.  - 
With  any  given  magnifier,  the 
size  of  the  field,  that  is  the 
area  which   can   be  seen,   is 
larger  with  the  eye  near  the 


FJG.  15-16.  VIRTUAL  IMAGE  WITH  THE 
OBJECT  NEAR  TO  AND  FAR  FROM  THE  PRIN- 
CIPAL Focus. 

/>  />  />  /  The  principal  foci  on  the  axis 
above  and  below  the  lens. 

Lc,  Lc  The  same  lens  to  show  the  dif- 
ference in  size  of  the  virtual  image  with 
the  object  near  to  and  far  from  the  princi- 
pal focus. 

ep     The  eye-point. 

A  B,  A'  B'  The  object  and  its  erect 
virtual  image. 


magnifier. 

Demonstrate  this  by  hold- 
ing the  eye  10  to  20  cm.  above 
the  tripod  magnifier  and^  noting  the  number  of  letters  or  words  which 
can  be  seen.  Then  lower  the  head  till  the  eye  is  only  2  to  5  cm.  from 
the  magnifier,  and  again  note  the  number  of  letters  or  words  which 
can  be  seen.  It  will  be  necessary  to  focus  the  magnifier  for  each 
position  of  the  eye. 


i6 


COMPOUND  MICROSCOPE  AND  ITS  PARTS 


[CH.  I 


17.     TRIPOD  MAGNIFIER. 


§  16.     Mounting  of  simple  microscopes.  —  Magnifiers  are  arranged 
in  mountings  to  be  held  in  the  hand;   for  example,  reading  glasses 

and  pocket  magnifiers.  The  tripod 
magnifier  (fig.  17)  may  be  held  in  the 
hand  or  supported  by  its  legs  over  the 
object  to  be  seen.  Sometimes  there  is 
a  special  support  with  arrangements 
for  focusing  as  well  as  holding  the 
magnifier  in  any  desired  position  (fig. 
19).  This  arrangement  is  especially 
desirable  when  magnifiers  are  used  for 
dissection.  For  the  purposes  of  dis- 
section and  examining  objects  under 
a  small  magnification,  binocular  ar- 
rangements like  spectacles  are  very 
convenient,  as  one  can  move  the  head  and  bring  the  object  into  view 
at  wiU  (§  145). 

COMPOUND  MICROSCOPE 

§  17.    This,  as  shown  in  fig.  3  and  20,  and  explained  above,  aids 
the  eye  in  obtaining  an  enlarged  retinal  image  by  two  steps,  viz.  the 

formation  of  a  large  real  image  by 

the  objective  and  a  retinal  image 
of  this  real  image  by  means  of  the 
microscope  ocular,  and  the  cornea 
and  crystalline  lens  of  the  eye,  the 
ocular  acting  in  general  like  a  simple 
microscope  (§  3). 

For  holding  the  objective  and  oc- 
ular and  focusing  the  microscope, 
there  are  a  number  of  mechanical 
arrangements  necessary.  For  illu- 
minating the  object  there  is  usually 
a  mirror  and  often  a  condenser.  It 

is  customary  and  convenient  to  divide  the  parts  of  a  compound  micro- 
scope into  two  groups:  (i)  the  optical  parts,  and  (2)  the  mechan- 
ical parts  (fig.  25). 


FIG.  1 8.  TRIPOD  MAGNIFIER  WITH 
A  SECTION  REMOVED  TO  SHOW  THE 
Two  COMPONENT,  CONVEX  LENSES 
AND  INTERVENING  DIAPHRAGM. 


CH.I] 


OBJECTIVES  FOR  THE  MICROSCOPE 


OPTICAL  PARTS  OF  A  COMPOUND  MICROSCOPE 
§  18.     Objective.  —  This  is  a  lens,  or  combination  of  lenses,  which, 
under  the  proper  conditions,  produces  an  enlarged,  inverted  image 
of  some  object  (fig.  n,  20). 


FIG.  19.     ADJUSTABLE  LENS  HOLDER  WITH  JOINTS. 

Base     The  heavy  base  supporting  the  lens  holder. 

Coarse  Adjustment     The  rack  and  pinion  for  focusing  the  lens. 

Joint,  Joint     The  joints  enabling  one  to  put  the  lens  in  any  desired  position. 

Lens     This  is  held  in  a  spring  fork  or  in  a  socket. 

Practically  all  microscopic  objectives  are  composed  of  one  or  of 
several  combinations  of  lenses.  The  purpose  of  combining  the 
lenses  is  to  produce  an  image  as  nearly  as  possible  like  the  object 
itself,  by  doing  away  with  certain  defects  or  aberrations  inherent  in 
simple  lenses  (fig.  21). 


i8 


OBJECTIVES  FOR  THE  MICROSCOPE 


[CH.  I 


the 


Mirror     The  plane  and  concave  mirror 
object  to  the  microscope  and  to  the  eye. 

Object    The  object  to  be  seen. 

/    The  principal  focus  of  the 

Objective   The  lens  serving  to 
ject. 

A  xis  The  principal  optic  axis 
eye. 

r  im    The  real  image  formed 
above   the   principal  focus  (/) 

Ocular  The  convex  lens 
real  image. 

cr     The    cornea    of 

rs     The   refracting  sur- 

L     The  crystalline  lens 

ri  The  retinal  image; 
with  the  real  image  formed 
as  compared  with  the 

Virtual  Image  The 
jected  into  the  field  of 
250  millimeters. 

This    image    is    in- 
with    the    object,   but 
the  real  image  formed 
The  microscope  enables 
small  object  as  if  it 
the  size  of  the  vir- 
placed  at  a  distance 
from  the  eye. 


to  reflect  the  light  up  through  the 


objective. 

form  a  real  image  of  the  ob- 

of  the  microscope  and  cf  the 


by  the  objective;   it  is  just 
%  rs   of  the  ocular. 

aiding  the  eye  to  see  the 


eye. 

face  of  the  schematic  eye. 

of  the  eye. 

it  is  inverted  as  compared 

by  the  objective,  but  erect 
object. 

retinal  image  pro- 
vision at  a  distance  of 


verted    as    compared 
erect  as  compared  with 
by  the  objective  (rim). 
the  eye  to  look  at  the 
were  enlarged  to 
tual    image    and 
of  2  50  millimeters 


Virtual 

»-»->- 

FIG.  20.     COMPOUND  MICROSCOPE  WITH  PROJECTED  VIRTUAL  IMAGE. 


CH.  I]  OBJECTIVES  FOR  THE  MICROSCOPE  19 

OBJECTIVES  AND  THEIR  DESIGNATION 

§  19.  Equivalent  focus.  —  In  America,  England,  and  now  also 
on  the  Continent,  objectives  are  designated  by  their  equivalent  focal 
length.  This  length  is  given  either  in  inches  (usually  contracted  to 
in.)  or  in  millimeters  (mm.).  Thus:  An  objective  designated  TV  in. 
or  2  mm.  indicates  that  the  objective  produces  a  real  image  of  the 
same  size  as  is  produced  by  a  simple  converging  lens  whose  principal 
focal  distance  is  yV  inch  or  2  millimeters  (fig.  10).  An  objective 
marked  3  in.  or  75  mm.  produces  approximately  the  same  sized  real 
image  as  a  simple  converging  lens  of  3  inches  or  75  millimeters  focal 
length. 

As  in  microscopic  work  the  object  is  always  very  close  to  the  prin- 
cipal focal  plane,  the  magnification  of  the  image  is  very  approximately 
the  number  obtained  by  dividing  the  image  distance  (fig.  84)  by  the 
equivalent  focus  of  the  objective.  It  follows  from  this  that  the  less 
the  focal  distance  of  the  objective,  the  greater  is  the  size  of  the  real 
image,  as  the  image  distance  remains  constant.  For  example,  if  the 
image  distance  is  250  mm.,  the  real  image  of  a  2  mm.  objective  is  -f-, 
or  125  times  longer  than  the  object:  of  a  5  mm.  objective  it  is  2-^-=  50 
times  longer,  etc.,  i.e.,  in  these  examples  the  magnification  is  125  and 
50  respectively  (§  iga). 

§  19a.  Initial  magnification.  —  In  addition  to  the  equivalent  focus,  some 
modern  objectives  are  marked  with  their  so-called  initial  magnification.  By 
this  is  meant  the  magnifying  power  of  the  objective  alone  at  some  standard 
image  distance.  For  example,  the  initial  magnification  of  the  Zeiss  2  mm. 
apochromatic  objective  is  given  as  125.  That  is,  the  image  distance  is  taken 
as  250  mm.  (-|-  =  125).  With  some  opticians  (Spencer  Lens  Co.,  Bausch 
&  Lomb  Optical  Co.)  the  initial  magnification  is  that  number  which  multi- 
plied by  the  power  of  the  ocular  gives  the  final  magnification  of  the  entire  micro- 
scope (tube-length  160  mm.,  projection  distance  of  the  virtual  image  by  the 
eye,  250  mm.).  If  one  multiplies  the  initial  magnification  by  the  equivalent 
focus  in  a  list  of  these  objectives,  the  image  distance  of  the  real  image  of  the 
objective  will  be  found  to  vary  from  160  to  180  mm.  That  is,  the  image 
distance  divided  by  the  equivalent  focus  would  give  the  initial  magnification 
listed,  only  by  varying  the  image  distance. 

§  20.  Numbering  or  lettering  objectives.  —  Instead  of  designating 
objectives  by  their  equivalent  focus,  many  Continental  opticians 
use  letters  or  figures  for  this  purpose;  in  most  cases,  however,  the 
equivalent  focus  is  also  given.  With  this  method  the  smaller  the 


20 


OBJECTIVES  FOR  THE  MICROSCOPE 


[Cn.  I 


number,  or  the  earlier  in  the  alphabet  the  letter,  the  lower  is  the 
power  of  the  objective.     This  method  is  entirely  arbitrary  and  does 


FIG.  21  A.     Low  OBJECTIVE  IN  SECTION. 

Axis     The  principal  optic  axis  of  the  objective. 
//     The  front  lens  of  the  objective. 

be    The  back  combination  composed  of  a  concave  and  a  convex  lens. 
Stage     The  stage  of  the  microscope  in  section. 

Mirror     The  mirror  is  above  the  stage  in  this  case  and  reflects  light  down 
upon  the  object. 

rl     Reflected  light  from  the  object. 
si     The  glass  slide. 
sp     The  specimen  on  the  slide. 
eg     Cover-glass  over  the  specimen. 

FIG.  21  B.     HIGH  POWER  OBJECTIVE  IN  SECTION. 

Axis     The  principal  optic  axis  of  the  objective. 

be     Back  combination  of  a  double  convex  and  a  plano-concave  lens. 

me     Middle  lens  combination. 

//     Front  lens  of  the  objective. 

eg,  sp,  si     The  cover-glass,  specimen,  and  slide. 

Stage     The  stage  of  the  microscope  in  section. 

Mirror     The  mirror  reflecting  parallel  rays  up  through  the  specimen. 

FIG.  21  C.     HIGH-POWER  OBJECTIVE  OF  FOUR  COMBINATIONS. 

j  The  front  lens. 

2,  3,  4     The  three  combinations  of  lenses,  the  back  combination  (4)  com- 
posed of  three  lenses. 

not,  like  the  one  above,  give  direct  information  concerning  the  ob- 
jective. 


CH.  I]  OBJECTIVES  FOR  THE  MICROSCOPE  21 

§  21.  Names  applied  to  parts  of  objectives.  —  As  objectives  have 
usually  two  or  more  combinations  of  lenses  (fig.  21  A-C)  it  is  con- 
venient to  have  a  name  for  each  combination. 

(1)  Front  combination.    This  is  the  part  of  the  objective  nearest 
the  object. 

(2)  Back  combination.    The  combination  of  lenses  farthest  above 
the  object,  and,  hence,  nearest  the  ocular. 

(3)  Intermediate   or   middle   combination.     The   lenses   between 
the  front  and  back  lenses.     Sometimes  there  are  two  or  more  inter- 
mediate combinations  (fig.  21  C). 

KINDS  or  OBJECTIVES 

Depending  on  their  construction  or  manner  of  use,  objectives  have 
received  special  designations  or  names. 

§  22.  Dry  objectives.  —  These  are  objectives  in  which  air  is  be- 
tween the  objective  and  the  object  or  cover-glass  (fig.  34). 

§  23.  Immersion  objectives.  —  With  these  there  is  some  liquid 
between  the  front  of  the  objective  and  the  object  or  the  cover-glass 
(fig.  21  B).  Immersion  objectives  are  usually  designated  by  the  name 
of  the  liquid  used. 

§  24.  Water  immersion  objectives.  —  With  these  there  is  water 
between  the  cover-glass  or  the  object  and  the  front  lens. 

§  25.  Homogeneous  or  oil  immersion  objectives.  —  The  immersion 
liquid  in  such  objectives  has  the  same  refractive  index  (see  Ch.  IX) 
as  glass,  hence  the  light  suffers  no  refraction  in  passing  from  the  glass 
slide  and  cover-glass  into  the  immersing  liquid,  and  from  that  into  the 
objective.  As  the  liquid  used  with  these  objectives  is  nearly  always 
thickened  cedar-wood  oil,  they  are  more  frequently  called  oil  immer- 
sion than  homogeneous  immersion  objectives. 

§  26.  Achromatic  objectives.  —  These  are  objectives  in  which 
the  image  is  practically  free  from  rainbow  colors.  They  are  com- 
posed of  one  or  more  combinations  of  convex  and  of  concave  lenses 
(see  Ch.  IX,  under  chromatic  aberration).  All  good  microscope 
objectives  are  achromatic. 

§  27.  Aplanatic  objectives,  etc.  —  These  are  objectives  or  other 
pieces  of  optical  arjparatus  (oculars,  illuminators,  etc.)  in  which  the 


22  OBJECTIVES  FOR  THE  MICROSCOPE  [Cn.  I 

spherical  distortion  is  wholly  or  nearly  eliminated,  and  the  curvatures 
are  so  made  that  the  central  and  marginal  parts  of  the  objective  focus 
rays  at  the  same  point  or  level.  Such  pieces  of  apparatus  are  usually 
achromatic  also. 

§  28.  Apochromatic  objectives.  —  By  this  is  meant  objectives  in 
which  by  means  of  special  forms  of  glass  and  a.  natural  mineral  (Cal- 
cium fluorid,  Fluorite,  Fluor-spar)  the  color  and  the  spherical  correc- 
tions have  been  made  especially  perfect,  that  is,  rays  of  three  spectral 
colors  are  combined  into  one  focus  instead  of  rays  of  two  colors  as 
with  the  ordinary  achromatic  objectives. 

§  29.  Semi-apochromatic,  parachromatic,  pantachromatic  objec- 
tives are  trade  names  for  those  containing  one  or  more  lenses  of  the 
new  forms  of  glass  and  are  said  to  approximate  more  closely  with 
the  apochromatic  than  with  the  older  achromatic  objectives. 

§  30.  Non-adjustable  or  unadjustable  objectives.  —  Objectives 
in  which  the  lenses  or  lens  systems  are  permanently  fixed  in  their 
mounting  so  that  their  relative  position  always  remains  the  same. 
Lower  power  objectives  and  those  with  homogeneous  immersion  are 
mostly  non-adjustable.  For  beginners  and  those  unskilled  in  manipu- 
lating adjustable  objectives  (§31),  non-adjustable  ones  are  more 
satisfactory,  as  the  optician  has  put  the  lenses  in  such  a  position  that 
the  most  satisfactory  results  may  be  obtained  when  the  proper  thick- 
ness of  cover-glass  and  tube-length  are  employed  (Ch.  IX). 

§  31.  Adjustable  objectives.  —  An  adjustable  objective  is  one  in 
which  the  distance  between  the  systems  of  lenses  (usually  the  front 
and  the  back  systems)  may  be  changed  by  the  observer  at  pleasure. 
The  object  of  this  adjustment  is  to  correct  or  compensate  for  the 
displacement  of  the  rays  of  light  produced  by  the  mounting  medium 
and  the  cover-glass  after  the  rays  have  left  the  object.  It  is  also  to 
compensate  for  variations  in  tube-length  (§  134).  As  the  displace- 
ment of  the  rays  by  the  cover-glass  is  the  most  constant  and  important, 
these  objectives  are  usually  designated  as  having  cover-glass  adjust- 
ment or  correction.  (See  also  practical  work  with  adjustable  objec- 
tives, §  134). 

§  32.  Variable  objective.  —  This  is  a  low  power  objective  of  36 
to  26  mm.  equivalent  focus,  depending  upon  the  position  of  the  com- 


CH.  I]  OCULARS  FOR  THE  MICROSCOPE  23 

binations.  By  means  of  a  screw  collar  the  combinations  may  be 
separated  or  brought  closer  together.  If  they  are  separated  the 
power  is  diminished;  and  if  brought  closer  together  the  power  is 
increased. 

§  33.  Illuminating  or  vertical  illuminating  objectives.  —  These 
are  designed  for  the  study  of  opaque  objects  with  good  reflecting 
surfaces,  like  the  rulings  on  metal  bars  and  broken  or  polished  and 
etched  surfaces  of  metals  employed  in  micro-metallography.  The 
light  enters  the  side  of  the  tube  or  objective  and  is  reflected  vertically 
downward  through  the  objective  and  thereby  is  concentrated  upon 
the  object.  The  object  reflects  part  of  the  light  back  into  the  micro- 
scope, thus  enabling  one  to  see  a  clear  image. 

§  34.  Low  and  high  objectives.  —  A  low  objective  is  one  that  mag- 
nifies relatively  little,  and  a  high  objective  is  one  that  magnifies  the 
real  image  greatly  (fig.  21  B,C).  By  looking  at  the  equivalent  focus 
of  an  objective  one  can,  of  course,  tell  very  precisely  concerning  its 
magnification  (§  19,  ipa),  but  it  is  also  very  convenient  to  judge  some- 
thing of  the  power  by  the  general  looks.  As  a  rough  statement  it 
may  be  said  that  a  high  power  usually  appears  more  elaborate  than 
a  low  power.  The  front  lens  is  usually  smaller,  and  the  whole  mount- 
ing is  usually  longer.  Conversely,  low  objectives  are  usually  shorter 
and  the  front  lens  larger  than  with  high  powers. 

OCULARS  AND  THEIR  DESIGNATION 

§  35.  An  ocular  or  eye-piece  for  the  microscope  consists  of  one 
or  more  converging  lenses  or  lens  systems  next  the  eye.  Its  main 
purpose  is  to  act  with  the  eye  as  a  magnifier  of  the  real  image  formed 
by  the  objective  (fig.  20).  Incidentally  the  ocular  also  serves  to  cor- 
rect some  of  the  defects  of  the  objective  (see  Ch.  IX). 

Oculars  may  be  divided  into  groups  according  to  their  construction 
or  action. 

§  36.  Positive  oculars.  —  These  have  the  real  image  of  the  objec- 
tive formed  below  all  the  lenses  of  the  ocular  (fig.  22  A,B). 

§  37.  Negative  oculars.  —  In  these  the  real  image  formed  by  the 
objective  is  between  the  lenses  (fig.  23,  24). 

In  a  negative  ocular  the  lower  or  field  lens  acts  with  the  objective 


OCULARS  FOR  THE  MICROSCOPE 


[Cn.  I 


to  form  the  real  image,  while  the  upper  or  eye  lens  acts  with  the  eye 
to  form  a  retinal  image  of  the  real  image  (fig.  23,  24). 


Eye- Point 


FIG.  22  A.     RAMSDEN  OCULAR  WITH  THE  REAL  IMAGE  BELOW  AND  THE 
EYE-POINT  ABOVE. 

Axis     The  principal  optic  axis  of  the  ocular. 

d,  ri  The  ocular  diaphragm  and  the  real  image  formed  by  the  objective 
below  the  lenses. 

Fl     The  field-lens  of  the  ocular. 

El     The  eye-lens. 

Eye-point  The  eye-point  in  section  and  in  face  view,  looking  at  the  upper 
end  of  the  ocular. 

FIG.  22  B.     POSITIVE  COMPENSATION  OCULAR. 

Axis     The  principal  optic  axis  of  the  ocular. 
d,  ri     The  ocular  diaphragm  and  the  real  image. 
FL     The  field  combination  composed  of  three  lenses. 
EL     The    eye-lens. 

Eye-point  The  eye-point  in  section  and  as  seen  by  looking  down  upon  the 
end  of  the  ocular. 


CH.  I] 


OCULARS  FOR  THE  MICROSCOPE 


Eye- Point 


Positive  and  negative  oculars  can  be  readily  distinguished  by  in- 
spection, as  the  ocular  diaphragm,  at  the  level  where  the  real  image  of 
the  objective  is  formed,  is  between 
the  lenses  of  the  negative  type,  and 
below  all  the  ocular  lenses  of  the 
positive  type  (fig.  22,  23). 

§  38.  Huygenian  ocular.  —  A 
negative  ocular  devised  by  the 
Dutch  astronomer  Huygens.  This 
is  the  most  common  ocular  used 
on  the  microscope,  and  consists  of 
a  plano-convex  field-lens  and  a 
similar,  but  higher  power,  eye-lens, 
the  convex  surfaces  of  both  facing 
downward  (fig.  23,  24).  Theoret- 
ically the  focal  length  of  the  field- 
lens  is  about  three  times  that  of 
the  eye-lens,  but  in  practice  the 
ratio  varies  with  the  power,  being 
i  to  1.5  or  i  to  2  with  low  powers 
and  nearer  i  to  3  with  the  high 
powers.  The  ocular  diaphragm  is 
placed  approximately  at  the  focus 
of  the  eye-lens. 

§  39.  Ramsden  ocular.  —  This 
is  a  positive  ocular  composed  of 
two  plano-convex  lenses  with  the 
convex  faces  turned  toward  each 
other,  and  so  arranged  that  the  real 
image  is  formed  below  both  lenses 
(fig.  22  A),  not  between  them,  as 
with  the  Huygenian  ocular.  In  the 
best  modern  forms  of  Ramsden  oc- 
ular the  simple  lenses  are  not  used,  but  achromatic  combinations  (see 
Ch.  IX  for  further  discussion).  The  Ramsden  form  is  often  used  for 
ocular  micrometers  (§  243). 


FIG.  23.     LOW-POWER    HUYGEXIAN 
OCULAR  IN  SECTION. 

Axis  The  principal  optic  axis  of 
the  ocular. 

FL     Field-lens  of  the  ocular. 

d,  ri  Diaphragm  and  real  image 
between  the  ocular  lenses. 

EL     Eye-lens  of  the  ocular. 

Eye-Point  The  eye-point  seen  in 
section  and  by  looking  down  upon 
the  end  of  the  ocular. 


26 


OCULARS  FOR  THE  MICROSCOPE 


.  I 


Eye- Point 


§  40.  Compensating  oculars.  —  These  are  either  positive  or  nega- 
tive oculars  chromatically  overcorrected  to  compensate  for  and 
correct  the  residual  color  defects  in  the  extra-axial  portion  of  the 

visual  field  due  to  the  non- 
achromatic  front  lens  of  the 
objective  (fig.  22  B).  They  are 
regularly  used  with  apochro- 
matic  objectives,  and  may  be 
used  to  advantage  with  high- 
angled  objectives  of  the  ordinary 
type  (see  further  in  Ch.  IX). 


POWER  OF  OCULARS 
As  oculars  of  all  kinds  are 
made  in  different  magnifying 
powers,  there  is  needed  some 
form  of  designation.  Many 
different  ways  of  designation 
have  been  used,  as  lettering, 
numbering,  giving  the  equiva- 
lent focus,  etc. 

§  41.  Lettering  or  number- 
ing. — This  is  a  purely  arbitrary 
form  of  designation,  but  prac- 
tically all  of  the  opticians 
adopted  the  rule  that  the  lower 
power  should  be  designated  by 
the  first  letter  of  the  alphabet, 
or  No.  i,  and  that  the  succeed- 
ing letters  or  numerals  should 


FIG.  24. 


HIGH-POWER  HUYGENIAN 
OCULAR. 


Axis     The  principal  optic  axis. 

FL     Field-lens. 

d,  ri  The  diaphragm  and  real  image 
between  the  ocular  lenses. 

EL     Eye-lens. 

Eye-point  The  eye-point  in  section 
and  face  view,  looking  down  upon  the 
upper  end  of  the  ocular. 


indicate  progressive  increase  in  power,  although  there  was  no  general 
agreement  as  to  the  exact  amount  of  that  increase.  Occasionally 
one  still  meets  with  oculars  lettered  A,  B,  C,  D,  or  numbered  i,  2,  3, 
4,  etc.  In  any  given  make  of  ocular  one  has  simply  to  remember 
that  the  earlier  the  letter  or  the  smaller  the  number,  the  lower  is  the 
power. 


CH.  I] 


OCULARS  FOR  THE  MICROSCOPE 


27 


OCULAR 


§  42.  Equivalent  focus.  —  Some  opticians  give  the  equivalent 
focus  of  the  ocular  as  with  objectives  (§  19) ;  then  the  user  can  select 
with  the  same  certainty  as  with 
objectives. 

§  43.  Magnification  of  ocu- 
lars. — The  most  recent  method 
is  to  mark  upon  the  ocular  the 
increase  it  gives  in  magnifica- 
tion to  the  objective  ($x,  lox, 
etc.).  If,  for  example,  the  real 
image  formed  by  the  objective 
is  10  times  larger  than  the  ob- 
ject, and  this  real  image  is  mag- 
nified 5  times  by  the  ocular,  the 
total  magnification  of  the  micro- 
scope is  50.  If  the  ocular  mag- 
nified 10,  then  the  final  image 
would  be  100  times  the  size  of 
the  object,  etc.  By  this  method 
the  part  done  by  the  ocular  can 
be  seen  by  inspecting  the  ocu- 
lar. (For  the  method  of  de- 
termining the  magnification  of 

the  ocular,  etc.,  see  Ch.  IX.)         FlG-  25-    LABORATORY  COMPOUND  MI- 
CROSCOPE WITH  THE  PARTS  NAMED. 


OBJECTIVE 


ENSER 


The  power  of  the  ocular  is 
also  indicated  by  the  appear- 
ance. A  long  ocular  in  which 
the  space  between  the  eye-lens 
and  field-lens  is  considerable, 
and  the  eye-lens  is  relatively 
large  is  usually  a  low  power 

(fig.  23).     If  the  ocular  is  short  and  the  eye-lens  relatively  small,  the 
ocular  has  a  relatively  high  power  (fig.  24). 

For  the  mechanical  parts  of  the  microscope  see  fig.  25,  and  §  164- 
166). 


Mirror,  Condenser,  Objective,  Ocular 
The  optical  parts  of  the  microscope 

Tube-length  This  is  the  space  between 
the  insertion  of  the  objective  below  and 
that  of  the  ocular  above.  It  is  most  com- 
monly 1 60  millimeters. 

Mechanical  parts  These  are  named  in 
order  from  the  base. 


28         PUTTING  OBJECTIVES  AND  OCULARS  IN  POSITION     [Cn.  I 


EXPERIMENTS  WITH  THE  COMPOUND  MICROSCOPE 
§  44.     Putting  an  objective  in  position  and  removing  it.  —  Elevate 
the  tube  of  the  microscope  by  means  of  the  coarse  adjustment  (fig. 

25)  so  that  there  may  be 
plenty  of  room  between 
its  front  or  lower  end  and 
the  stage.  Grasp  the  ob- 
jective lightly  near  its 
lower  end  with  two  fingers 
of  the  left  hand,  and  hold 
it  against  the  nut  at  the 
lower  end  of  the  tube  or 
the  revolving  nose-piece 
(fig.  26-28).  With  two 
fingers  of  the  right  hand 


ring  near  the  back  or  up- 
per end  of  the  objective  and  screw  it  into  the  tube  of  the  microscope 
or  nose-piece.     Reverse  this  operation  for  removing  the  objective. 
By  following  this  method 
the  danger  of  dropping  the 
objective  will  be  avoided. 
§  45.    Putting  an  ocular 
in  position  and  removing 
it.  —  Elevate  the  body  of 
the  microscope  with   the 
coarse  adjustment  so  that 
the  objective  will  be  2  cm. 
or  more  from  the  object, 
grasp  the  ocular   by  the 
milled  ring  next  the  eye- 
lens  (fig.  25)  and  the  coarse 
adjustment  or  the  tube  of 
the  microscope  an.d  gently  FJG   ^     TRIpLE  NosE.PIECE  mTH  THE  THREE 
force  the  ocular  into  posi-  OBJECTIVES  IN  POSITION. 


CH.  I] 


FIELD  OF  THE  MICROSCOPE 


29 


tion.  In  removing  the  ocular,  reverse  the  operation.  If  the  above 
precautions  are  not  taken,  and  the  oculars  fit  snugly,  there  is  danger 
in  inserting  them  of  forc- 
ing the  tube  of  the  micro- 
scope downward  and  the 
objective  upon  the  object. 
§  46.  Putting  an  object 
under  the  microscope.  — 
This  is  so  placing  an  object 
under  the  simple  micro- 
scope, or  on  the  stage  of 
the  compound  microscope, 
that  it  will  be  in  the  field 
of  view  when  the  micro- 
scope is  in  focus  (§  47,  69, 


FIG.  28.     QUADRUPLE  NOSE-PIECE  WITH  THE 
FOUR  OBJECTIVES  IN  PLACE. 


fig-  63). 

With  low  powers,  it  is  not  difficult  to  get  an  object  under  the  micro- 
scope. The  difficulty  increases,  however,  with  the  power  of  the  micro- 
scope and  the  smallness  of  the 
object.  It  is  usually  necessary 
to  move  the  object  in  various 
directions  while  looking  into  the 
microscope,  in  order  to  get  it 
into  the  field.  Time  is  usually 
saved  by  getting  the  object  in 
the  center  of  the  field  with  a 
low  objective  before  putting  the 
high  objective  in  position.  This 


B 


09 

32 

FIG.  29. 


16 


8 


4      2 


MICROSCOPIC  FIELD  WITH  AND    js  greatly  facilitated  by  using  a 
WITHOUT  OCULARS. 

nose-piece,  or  revolver  (fig.  26- 

28). 

§  47.  Field  or  field  of  view 
of  a  microscope.  —  This  is  the 
area  visible  through  a  micro- 


A  The  field  of  the  2.  4.  8.  16  and  32 
mm.  objectives  without  an  ocular. 

B  Field  of  the  same  objectives  with  a 
5x  ocular. 

C  Field  of  the  same  objectives  with  a 
i  ox  ocular. 


3216,8,4,2    Equivalent  focus  of    SCOpe  when  it  is  in  focus.    When 
the  different  objectives  whose  fields  are 
shown.  properly  lighted  and  there  is  no 


FIELD  OF  A  MICROSCOPE 


CCH.  I 


object  under  the  microscope,  the  field  appears  as  a  disc  of  light.  When 
examining  an  object  it  appears  within  the  light  circle,  and  by  moving 
the  object,  if  it  is  of  sufficient  size,  different  parts  are  brought  succes- 
sively into  the  field  of  view. 

In  general,  the  greater  the  magnification  of  the  entire  microscope, 
whether  the  magnification  is  produced  mainly  by  the  objective,  the 
ocular,  or  by  increasing  the  tube-length,  or  by  a  combination  of  all 
three  (§  235),  the  smaller  is  the  field. 

The  size  of  the  field  is  also  dependent,  in  part,  without  regard  to 
magnification,  upon  the  size  of  the  opening  in  the  ocular  diaphragm. 
Some  oculars,  as  the  orthoscopic  and  periscopic,  are  so  constructed 
as  to  eliminate  the  ocular  diaphragm,  and  in  consequence,  although 
this  is  not  the  sole  cause,  the  field  is  considerably  increased. 

§  48,  Measuring  the  size  of  the  field.  —  Use  a  stage  micrometer 
(fig.  80)  as  object,  and  read  off  the  number  of  spaces  required  to  meas- 
ure the  diameter  of  the  light  disc  as  seen  in  the  microscope.  Use 
first  a  low  objective  (16  mm.)  and  a  low  ocular  (4x  or  5x),  then  use  the 
higher  ocular  (8x  or  lox).  Do  the  same  with  the  4  or  8  mm.  objective 
and  the  two  oculars.  Make  a  table  giving  the  diameter  of  the  field 
in  each  case  and  compare  with  the  accompanying  table.  The  tube- 
length  (fig.  25)  should  be  160  mm.  when  making  the  measurements. 
To  see  the  effect  of  lengthening  the  tube,  pull  it  out  till  the  tube-length 
is  200  mm.  and  note  the  effect  on  the  size  of  the  field  with  one  objective 
and  the  two  oculars.  (The  longer  the  tube  the  smaller  the  field). 

§  49.  Table  showing  the  actual  size  of  the  field  of  view  of  various  objec- 
tives and  oculars  with  a  tube-length  of  160  mm. 


Objective 

5* 
Ocular 

Diameter  of 
Field  in  mm. 

I  OX 

Ocular 

Diameter  of 
Field  in  mm. 

40  mm. 

7-3 

4-85 

32  mm. 

5-3 

3-7 

25  mm. 

3-5 

2-55 

16  mm. 

2.15 

i-55 

12  mm. 

1.2 

0.85 

8  mm. 

O.Q7 

0.69 

4  mm. 

0.44 

0.31 

3  mm. 

0.31 

0.225 

2  mm. 

O.2I 

0.155 

CH.  I]  FUNCTION  OF  AN  OBJECTIVE  31 

FUNCTION  OF  AN  OBJECTIVE 

§  50.  Put  a  50  mm.  objective  on  the  microscope,  or  screw  off  the 
front  combination  of  a  16  mm.,  and  put  the  back  combination  on  the 
microscope  for  a  low  objective. 

Place  some  printed  letters  or  figures  under  the  microscope,  and 
light  well.  In  place  of  an  ocular  put  a  screen  of  ground-glass,  or  a 
piece  of  lens  paper,  over  the  upper  end  of  the  tube  of  the  microscope. 

Lower  the  tube  of  the  microscope  by  means  of  the  coarse  adjust- 
ment until  the  objective  is  within  2  to  3  cm.  of  the  object  on  the  stage. 
Look  at  the  screen  on  the  top  of  the  tube,  holding  the  head  about  as 
far  from  it  as  for  ordinary  reading,  and  slowly  elevate  the  tube  by 
means  of  the  coarse  adjustment  until  the  image  of  the  letters  appears 
on  the  screen. 

The  image  can  be  more  clearly  seen  if  the  object  is  in  a  strong  light 
and  the  screen  in  a  moderate  light,  i.e.,  if  the  top  of  the  microscope  is 
shaded. 

The  letters  will  appear  as  if  printed  on  the  ground-glass  or  paper, 
but  will  be  inverted. 

If  the  objective  is  not  raised  sufficiently,  and  the  head  is  held 
too  near  the  microscope,  the  objective  will  act  as  a  simple  microscope. 
If  the  letters  are  erect,  and  appear  to  be  down  in  the  microscope 
and  not  on  the  screen,  hold  the  head  farther  from  it,  shade  the  screen, 
and  raise  the  tube  of  the  microscope  until  the  letters  do  appear  on 
the  ground-glass. 

§  60a.  Ground-glass  may  be  very  easily  prepared  by  placing  some  fine 
emery  or  carborundum  between  two  pieces  of  glass,  wetting  it  with  water,  and 
then  rubbing  the  glasses  together  for  a  few  minutes.  If  the  glass  becomes  too 
opaque,  it  may  be  rendered  more  translucent  by  rubbing  some  oil  upon  it. 

§  51.  Aerial  image.  —  After  seeing  the  real  image  on  the  ground- 
glass  or  paper,  use  the  lens  paper  over  about  half  of  the  opening  of 
the  tube  of  the  microscope.  Hold  the  eye  about  250  mm.  from  the 
microscope  as  before  and  shade  the  top  of  the  tube  by  holding  the  hand 
between  it  and  the  light,  or  in  some  other  way.  The  real  image  can 
be  seen  in  part  as  if  on  the  paper  and  in  part  in  the  air.  Move  the 
paper  so  that  the  image  of  half  a  letter  will  be  on  the  paper  and  half 


32  FUNCTION  OF  AN  OCULAR  c        [Cn.  I 

in  the  air.  Another  striking  experiment  is  to  have  a  small  hole  in 
the  paper  placed  over  the  center  of  the  tube  opening,  then  if  a  printed 
word  extends  entirely  across  the  diameter  of  the  tube,  its  central 
part  may  be  seen  in  the  air,  the  lateral  parts  on  the  paper.  The  ad- 
vantage of  the  paper  over  part  of  the  opening  is  to  enable  one  to  accom- 
modate the  eyes  for  the  right  distance.  If  the  paper  is  absent  the  eyes 
adjust  themselves  for  the  light  circle  at  the  back  of  the  objective,  and 
the  aerial  image  appears  low  in  the  tube.  Furthermore  it  is  more 
difficult  to  see  the  aerial  image  in  space  than  to  see  the  image  on  the 
ground-glass  or  paper,  for  the  eye  must  be  held  in  the  right  position 
to  receive  the  rays  projected  from  the  real  image,  while  the  granular 
surface  of  the  glass  and  the  delicate  fibers  of  the  paper  reflect  the  rays 
irregularly,  so  that  the  image  may  be  seen  at  almost  any  angle,  as  if 
the  letters  were  actually  printed  on  the  paper  or  glass. 

§  52.  The  function  of  an  objective,  as  seen  from  these  experiments, 
is  to  form  an  enlarged,  inverted,  real  image  of  an  object,  this  image 
being  formed  on  the  opposite  side  of  the  objective  from  the  object 
(fig.  13,  20). 

FUNCTION  OF  AN  OCULAR 

§  53.  Using  the  same  objective  as  for  §  50,  get  as  clear  an  image 
of  the  letters  as  possible  on  the  lens  paper  or  ground-glass  screen. 
Look  at  the  image  with  a  simple  microscope  (fig.  17),  as  if  the  image 
were  an  object. 

Observe  that  the  image  seen  through  the  simple  microscope  is 
merely  an  enlargement  of  the  one  on  the  screen,  and  that  the  letters 
remain  inverted.  Remove  the  screen  and  observe  the  aerial  image 
with  the  tripod  magnifier. 

§  54.  Put  a  4x  or  $x  ocular,  i.e.,  an  ocular  of  low  magnification  in 
position  (§  45).  Hold  the  eye  about  10  to  20  mm.  from  the  eye-lens 
and  look  into  the  microscope.  The  letters  will  appear  as  when  the 
simple  microscope  was  used  (see  above) ;  the  image  will  become  more 
distinct  by  slightly  raising  the  tube  of  the  microscope  with  the  coarse 
adjustment. 

§  55.  The  function  of  the  ocular,  as  seen  from  the  above,  is  that 
of  a  simple  microscope,  viz.  it  magnifies  the  real  image  formed  by 


CH.  I]  EYE-POINT  OF  AN  OCULAR  33 

the  objective  as  if  that  image  were  an  object.  Compare  the  image 
formed  by  the  ocular  (fig.  3,  20)  and  that  formed  by  a  simple  micro- 
scope (fig.  2,  6). 

It  should  be  borne  in  mind,  however,  that  the  rays  from  an  object 
as  usually  examined  with  a  simple  microscope  extend  from  the  object 
in  all  directions,  and  no  matter  at  what  angle  the  simple  microscope 
is  held,  provided  it  is  sufficiently  near  and  points  toward  the  object, 
an  image  may  be  seen.  The  rays  from  a  real  image,  however,  are 
continued  in  certain  definite  lines  and  not  in  all  directions;  hence,  in 
order  to  see  this  aerial  image  with  an  ocular  or  simple  microscope,  or 
in  order  to  see  the  aerial  image  with  the  unaided  eye,  the  simple  micro- 
scope, ocular,  or  eye  must  be  in  the  path  of  the  rays  (fig.  2-3). 

§  56.  The  field-lens  of  a  Huygenian  ocular  makes  the  real  image 
smaller  and  consequently  increases  the  size  of  the  field;  it  also  makes 
the  image  brighter  by  contracting  the  area  of  the  real  image  (fig.  23, 
24).  Demonstrate  this  by  screwing  off  the  field-lens  and  using 
the  eye-lens  alone  as  an  ocular,  refocusing  if  necessary.  Note  that 
the  image  is  bordered  by  a  colored  haze  (Ch.  IX). 

When  looking  into  the  ocular  with  the  field-lens  removed,  the  eye 
should  not  be  held  so  close  to  the  ocular,  as  the  eye-point  (fig.  23) 
is  considerably  farther  away  than  when  the  field-lens  is  in  place. 

§  57.  Eye-point.  —  This  is  in  the  plane  above  the  ocular  where 
the  emerging  rays  cross  (fig.  22-24).  If  the  eye  is  placed  at  this 
point  it  will  receive  the  greatest  number  of  rays  from  the  microscope 
and  thus  see  the  largest  field.  If  the  eye  is  too  far  from  or  too  near 
the  ocular,  part  of  the  rays  cannot  enter  the  pupil  of  the  eye  and  the 
microscopic  image  is  restricted. 

Demonstrate  the  eye-point  by  using  a  16  mm.  objective  and  a  4x 
or  5x  ocular.  Light  brightly  and  then  focus  the  microscope  on  some 
transparent  specimen.  Open  the  diaphragm  widely  so  that  the  entire 
aperture  of  the  objective  is  filled  with  light  (fig.  45).  Shade  the 
ocular  with  the  hand  or  a  screen  and  hold  above  the  eye-lens  a  piece 
of  ground-glass  or  of  the  lens  paper.  By  raising  and  lowering  the 
glass  or  paper  one  will  find  the  level  where  the  sharpest  and  brightest 
light  circle  is  located.  The  height  varies  with  different  oculars.  Now 
use  the  tripod  or  other  magnifier  and  look  at  the  eye-point.  It  is 


34  ERECT  AND  INVERTED  IMAGES  [CH.  I 

really  the  image  of  the  aperture  of  the  objective,  and,  as  shown  later, 
the  study  of  this  image  enables  one  to  detect  lint  and  other  particles 
on  the  upper  lens  of  the  objective  (§  5ya). 

The  eye-point  is  also  known  as  the  Pupil  of  the  lens;  Ramsden 
Disc  or  Circle;  Lagrange  Disc. 

§  67a.  As  pointed  out  by  Wright  (p.  93),  a  study  of  the  eye-point  with  a 
magnifier  gives  very  definite  information  and  guidance  on  several  important 
points: 

(1)  The  aperture  of  the  light  in  the  objective,  and  hence  whether  the  dia- 
phragm of  the  condenser  is  opened  the  right  amount. 

(2)  The  centering  of  the  condenser. 

(3)  The  presence  of  dust  or  other  opacities  on  the  back  lens. 

(4)  The  partial  unsealing  of  any  of  the  objective  combinations. 

(5)  The  presence  of  air  bubbles  in  the  immersion  liquid. 

§  58.     Erect    and   inverted    images   with    the    microscope.  —  By 

glancing  at  fig.  2,  6  it  will  be  seen  that  with  the  simple  microscope 
the  retinal  image  is  inverted;  that  is,  the  arrow  is  turned  end  for  end. 
In  like  manner  the  retinal  image  of  any  object  seen  with  the  naked  eye 
is  also  inverted  (fig.  5). 

On  the  other  hand,  with  the  compound  microscope,  the  retinal  image 
is  erect  (fig.  3,  20);  that  is,  the  arrow  points  in  the  same  direction  as 
the  object.  This  is  because  the  eye  does  not  see  the  object  directly, 
but  the  real  image  formed  by  the  objective,  and  this  is  inverted. 
From  the  crossing  of  the  rays  on  entering  the  eye,  this  inverted  real 
image  is  reinverted,  and  thus  gives  an  erect  image  on  the  retina.  Now 
as  objects  or  their  images  do  not  seem  to  be  on  the  retinal  screen,  but 
out  in  space  in  the  direction  of  the  light  rays  entering  the  eye,  it  is 
very  evident  that  if  the  light  rays  are  traced  from  the  retinal  image  to 
the  object  or  to  a  virtual  image,  this  will  appear  to  be  erect  when  the 
image  on  the  retina  is  inverted,  and  it  will  appear  inverted  when  the 
retinal  image  is  erect,  because  of  the  crossing  of  the  rays  in  passing 
the  pupil  of  the  eye  (fig.  2,  3,  6,  20)  on  their  way  to  the  retinal  image, 
or  on  their  way  from  the  retinal  image  to  the  apparent  position  of  the 
object  or  the  virtual  image. 


CH.  I]  COLLATERAL  READING  35 

COLLATERAL  READING 

BEALE,  L.  S.  —  How  to  Work  with  the  Microscope. 
CARPENTER-DALLINGER.  —  The  Microscope  and  its  Revelations. 
CHAMOT,  E.  M.  —  Elementary  Chemical  Microscopy. 
HOWELL,  W.  H.  —  A  Text-book  of  Physiology. 

.SPITTA,  E.  J.  —  Microscopy,  the   Construction,  Theory  and  Use  of  the  Mi- 
croscope. 

WINSLOW,  C.  E.  A.  —  Elements  of  Applied  Microscopy. 
WRIGHT,  SIR  A.  E.  —  Principles  of  Microscopy. 
Journal  of  the  Royal  Microscopical  Society.     (See  the  Bibliography  at  the  end.) 


CHAPTER  II 

FOCUSING  THE  MICROSCOPE;  WORKING  DISTANCE;  LIGHTING 
WITHOUT  AND  WITH  A  CONDENSER;  ARTIFICIAL  DAY- 
LIGHT; DARK  GROUND  ILLUMINATION 

§  68.     Apparatus  and  material  for  Chapter  II. 

1.  Microscope  supplied  with  plane  7.  Stage  micrometer  (fig.  80). 
and  concave  mirror,  achromatic  and           8.   10%   solution   salicylic   acid   in 
Abbe     condensers,     dry,     adjustable       95%  alcohol;    cedar  oil. 

and    immersion    objectives,    oculars,  9.  Glass    slides    and    cover-glasses 

triple  nose-piece  (fig.  25).  (Ch.    X). 

2.  Lamp  or  lantern  for  microscopic  10.  Preparation    of    stained    bac- 
vvork  (fig.  37-38);  opaque  screen.  teria. 

3.  Homogeneous  immersion  liquid;  n.  Vial    of    equal    parts    olive    or 
xylene;   alcohol;   distilled   water.  cottonseed  oil,  or  liquid  vaseline  and 

4.  Mounted    preparation    of    fly's  xylene. 

wing;    lint;  samples  of  starch.  12.  Black  and  colored  ink;  pencils. 

5.  Simple  microscope;    steel  scale  13.  Gum    arabic    mucilage, 
ruled  in   \   mm.  14.  Dark         ground         condenser 

6.  Preparation      of       Pleurosigma  (§  125). 

(§  98,  115);   piece  of  black  velvet.  15.  Small  arc  lamp  (fig.  49). 

FOCUSING 

§  69.  Focusing  is  mutually  arranging  an  object  and  the  microscope 
so  that  a  clear  image  may  be  seen. 

With  a  simple  microscope  either  the  object  or  the  microscope  or 
both  may  be  moved  in  order  to  see  the  image  clearly,  but  with  the 
compound  microscope  the  object  more  conveniently  remains  sta- 
tionary on  the  stage,  and  the  tube  or  body  of  the  microscope  is  raised 
or  lowered  (fig.  25). 

In  general,  the  higher  the  power  of  the  whole  microscope,  whether 
simple  or  compound,  the  nearer  together  must  the  object  and  the 
objective  be  brought.  With  the  compound  microscope,  the  higher 
the  objective,  and  the  longer  the  tube  of  the  microscope,  the  nearer 
together  must  the  object  and  the  objective  be  brought.  If  the  oculars 
are  not  parfocal,  the  higher  the  magnification  of  the  ocular,  the  nearer 
must  the  object  and  objective  be  brought. 

36 


CH.  II]  FOCUSING  THE  MICROSCOPE  37 

FOCUSING  EXPERIMENTS 

§  70.  Focusing  low  objectives.  —  Place  a  mounted  fly's  wing 
under  the  microscope;  put  the  16  mm.  objective  and  the  4x  or  5x 
ocular  in  position.  Select  the  proper  opening  in  the  diaphragm  and 
light  the  object  well  with  transmitted  light  (§  85). 

Hold  the  head  at  about  the  level  of  the  stage,  look  toward  the 
window,  and  between  the  object  and  the  front  of  the  objective;  with 
the  coarse  adjustment  lower  the  tube  until  the  objective  is  within 
about  half  a  centimeter  of  the  object.  Then  look  into  the  microscope 
and  slowly  elevate  the  tube  with  the  coarse  adjustment.  The  image 
will  appear  dimly  at  first,  but  will  become  very  distinct  by  raising  the 
tube  still  higher.  If  the  tube  is  raised  too  high  the  image  will  become 
indistinct,  and  finally  disappear.  It  will  again  appear  if  the  tube  is 
lowered  the  proper  distance. 

When  the  microscope  is  well  focused  try  both  the  concave  and  the 
plane  mirrors  in  various  positions  and  note  the  effect. 

Pull  out  the  draw- tube  4  to  6  cm.,  thus  lengthening  the  body  of 
the  microscope;  it  will  be  found  necessary  to  lower  the  tube  of  the 
microscope  somewhat  (for  reason,  see  fig.  83). 

§  71.  Pushing  in  the  draw-tube.  —  To  push  in  the  draw-tube, 
grasp  the  large  milled  ring  of  the  ocular  with  one  hand,  and  the  milled 
head  of  the  coarse  adjustment  with  the  other,  and  gradually  push 
the  draw-tube  into  the  tube.  If  this  were  done  without  these  pre- 
cautions the  objective  might  be  forced  against  the  object  and  the 
ocular  thrown  out  by  the  compressed  air. 

§  72.  Focusing  with  high  objectives.  —  Employ  the  same  object 
as  before,  elevate  the  tube  of  the  microscope  and,  if  no  revolving  nose- 
piece  is  present,  remove  the  16  mm.  objective  as  indicated.  Put  a 
4  mm.  or  higher  objective  in  place,  and  use  a  4x  or  5x  ocular. 

Light  well,  and  employ  the  proper  opening  in  the  diaphragm,  etc. 
(§  89).  Look  between  the  front  of  the  objective  .and  the  object  as 
before  (§  70),  and  lower  the  tube  with  the  coarse  adjustment  till  the 
objective  almost  touches  the  cover-glass  over  the  object.  Look  into 
the  microscope,  and  with  the  coarse  adjustment,  raise  the  tube  very 
slowly  until  the  image  begins  to  appear,  then  turn  the  milled  head  of 


38  PARFOCAL  OCULARS  AND  OBJECTIVES  [Cn.  H 

the  fine  adjustment  (fig.  25),  first  one  way  and  then  the  other,  until 
the  image  is  sharply  defined. 

In  practice  it  is  found  of  great  advantage  to  move  the  preparation 
slightly  while  focusing.  This  enables  one  to  determine  the  approach 
to  the  focal  point  either  from  the  shadow  or  the  color,  if  the  object  is 
colored.  With  high  powers  and  scattered  objects  there  might  be  no 
object  in  the  small  field  (§  47,  fig.  29  for  size  of  field).  By  moving 
the  preparation  an  object  will  be  moved  across  the  field  and  its  shadow 
gives  one  the  hint  that  the  objective  is  approaching  the  focal  point. 
It  is  sometimes  desirable  to  focus  on  the  edge  of  the  cement  ring  or  on 
the  little  ring  made  by  the  marker  (fig.  61). 

§  73.  Always  focus  up,  as  directed  above.  —  If  one  lowers  the 
tube  only  when  looking  at  the  end  of  the  objective  as  directed  above, 
there  will  be  no  danger  of  bringing  the  objective  in  contact  with  the 
object,  as  may  be  done  if  one  looks  into  the  microscope  and  focuses 
down. 

When  the  instrument  is  well  focused,  move  the  object  around  in 
order  to  bring  different  parts  into  the  field.  It  may  be  necessary  to 
refocus  with  the  fine  adjustment  every  time  a  different  part  is  brought 
into  the  field.  In  practical  work  one  hand  is  kept  on  the  fine  adjust- 
ment constantly,  and  the  focus  is  continually  varied. 

§  74.  Parfocal  oculars  and  focusing.  —  On  changing  the  oculars 
from  a  higher  to  a  lower  or  the  reverse  it  is  necessary  to  refocus  the 
microscope.  Formerly  the  change  in  focus  was  very  marked  in  chang- 
ing from  one  power  of  ocular  to  another,  but  since  Mr.  Pennock 
introduced  parfocal  oculars  (1881)  and  their  almost  universal  adoption 
since,  very  little  change  in  focus  is  necessary  in  passing  from  power  to 
power  of  ocular  (see  Ch.  IX). 

§  75.  Parfocal  objectives.  —  These  are  groups  of  objectives,  of 
different  power,  so  mounted  that  when  screwed  into  the  revolving 
nose-piece  of  the  microscope  very  little  change  in  focusing  is  necessary 
in  passing  from  objective  to  objective.  This  arrangement  of  objectives 
was  a  natural  outgrowth  from  the  parfocalization  of  the  oculars 

(§  74). 

In  case  the  objectives  are  not  nearly  enough  parfocal  so  that  the 
object  is  visible  in  turning  from  one  objective  to  another,  the  defect 


CH.  II]        WORKING  DISTANCE  WITH  THE  MICROSCOPE  39 

can  be  easily  corrected  by  getting  one  of  the  objectives  in  exact  focus 
and  then  turning  the  others  successively  into  place.  If  one  notes 
whether  it  is  necessary  to  focus  up,  then  it  will  be  known  that  the  ob- 
jective projects  too  far  down  toward  the  object;  if,  on  the  other  hand, 
one  must  focus  down,  then  the  objective  is  too  high  up.  To  correct 
this  lack  of  parfocalization  use  the  objective  which  projects  farthest 
toward  the  object  as  standard.  Focus  it  sharply  and  then  turn  another 
in  position.  Unscrew  this  slowly  until  the  image  is  also  sharp.  Now 
wind  a  thread  or  string  around  the  lower  end  of  the  objective  screw 
and  then  turn  it  in  place  and  slowly  screw  it  into  the  revolving  nose- 
piece  until  it  is  in  focus.  Proceed  with  all  until  the  entire  number 
are  in  focus  at  the  same  level.  With  parfocal  oculars  and  parfocal 
objectives  much  time  and  annoyance  is  saved,  for  one  can  see  the 
specimen  in  turning  from  power  to  power,  and  it  is  only  necessary  to 
make  a  small  focusing  adjustment  to  get  the  best  image. 

§  76.  Working  distance.  —  By  this  is  meant  the  space  between 
the  simple  microscope  and  the  .object,  or  between  the  front  lens  of 
the  compound  microscope  and  the  object,  when  the  microscope  is 
in  focus.  This  working  distance  is  always  considerably  less  than 
the  equivalent  focal  length  of  the  objective.  For  example,  the  front- 
lens  of  a  4  mm.  objective  would  not  be  4  millimeters  from  the 
object  when  the  microscope  is  in  focus,  but  considerably  less  than 
that  distance,  viz.  less  than  half  a  millimeter.  If  now  a  cover-glass 
of  half  a  millimeter  or  more  in  thickness  were  used  it  would  be  impos- 
sible to  get  the  4  mm.  objective  near  enough  the  object  to  get  it  in 
focus.  It  is  not  uncommon  for  students  to  put  their  microscopic 
specimens  on  the  stage  of  the  microscope  wrong  side  up.  Then  the 
thickness  of  the  slide  is  over  the  object.  With  low  powers  the  object 
can  still  be  put  in  focus;  but  not  with  high  powers,  as  the  working 
distance  is  not  great  enough.  See  also  aberrations  produced  by  the 
cover-glass  (fig.  51). 

§  77.  Free  working  distance.  —  (i)  Where  no  cover-glass  is  used 
this  is  the  distance  between  the  front  of  the  magnifier  or  the  front 
lens  mount  of  the  objective  and  the  object  (fig.  30). 

(2)  If  a  cover-glass  is  used,  it  is  the  distance  between  the  upper 
surface  of  the  cover-glass  and  the  magnifier  or  objective  when  the 


40  WORKING  DISTANCE  WITH  THE  MICROSCOPE       [Cn.  II 

microscope  is  in  focus  (fig.  31,  34).  Strictly  speaking,  it  is  the  dis- 
tance between  the  objective  front  and  the  upper  surface  of  a  cover- 
glass  of  the  exact  thickness  for  which  the  objective  is  corrected 


FIG.  30,  31,  32.     WORKING  DISTANCE  AND  THE  COVER-GLASS. 

Slide     The  glass  slide  upon  which  the  object  is  mounted. 

A     Working  distance  with  an  uncovered  object. 

B  Working  distance  when  a  cover-glass  is  used  and  the  object  is  in  contact 
with  the  cover-glass.  The  object  represented  by  the  solid  black  oblong  ap- 
pears to  be  elevated  one  third  the  thickness  of  the  cover  to  the  level  Obj.,  where 
it  is  represented  by  dots. 

The  objective  is  elevated  corresponding  to  the  apparent  elevation  of  the 
object. 

C  Working  distance  when  a  cover-glass  is  used  and  the  objects  are  dis- 
tributed in  a  stratum  of  Canada  balsam. 

It  is  evident  from  this  figure  why  the  focus  must  be  different  for  objects 
at  different  depths  in  the  balsam. 


As  the  working  distance  of  an  objective  is  practically  always  less 
than  its  equivalent  focus,  one  must  take  care  to  use  cover-glasses  thin 
enough  so  that  any  suitable  objective  can  be  used  for  studying  the 
specimen.  Furthermore,  as  microscopic  specimens  have  considerable 
thickness,  the  cover-glass  should  be  thin  enough  so  that  the  objective 
can  be  lowered  sufficiently  to  enable  one  to  bring  the  lower  strata 
of  the  specimen  in  focus  without  bringing  the  objective  front  in  con- 
tact with  the  upper  surface  of  the  cover-glass  (fig.  32). 


CH.  II]       WORKING  DISTANCE  WITH  THE  MICROSCOPE  41 

DETERMINATION  OF  WORKING  DISTANCE 

§  78.  Working  distance,  no  cover. — As  stated  in  §  77,  this  is 
the  distance  between  the  front  lens  or  mounting  of  the  front  lens  of 
the  objective  and  the  object  when  the  objective  is  in  focus.  It  is 
always  less  than  the  equivalent  focal  length  of  the  objective. 

Make  a  wooden  wedge  10  cm.  long  which  shall  be  exceedingly  thin 
at  one  end  and  about  20  mm.  thick  at  the  other.  Place  a  slide  on  the 
stage  and  some  dust  or  an  ink  or  pencil  mark  on  the  slide.  Do  not 
use  a  cover-glass.  Use  a  16  mm.  objective  and  focus  the  dust  or  mark 
carefully,  and  when  the  objective  is  in  focus  push  the  wedge  between 
the  objective  and  slide  until  it  touches  the  objective.  Mark  the 
place  of  contact  with  a  pencil  and  then  measure  the  thickness  of  the 
wedge  with  a  rule  opposite  the  point  of  contact.  This  thickness  will 
represent  very  closely  the  working  distance.  For  measuring  the 
thickness  of  the  wedge  at  the  point  of  contact  for  the  high  objective 
use  a  steel  scale  ruled  in  £  mm.  and  the  tripod  magnifier  to  see  the 
divisions.  Or  one  may  use  a  cover-glass  measurer  (Ch.  X)  for  deter- 
mining the  thickness  of  the  wedge. 

For  the  higher  powers,  if  one  has  a  microscope  in  which  the  fine 
adjustment  is  graduated,  the  working  distance  may  be  readily  deter- 
mined as  follows: 

Use  the  marked  slide  as  above.  Get  the  dust  or  mark  in  focus, 
then  lower  the  tube  of  the  microscope  until  the  front  of  the  objective 
just  touches  the  slide.  Note  the  position  of  the  micrometer  screw  and 
slowly  focus  up  with  the  fine  adjustment  until  the  dust  or  mark  is 
again  in  focus.  By  noting  the  total  and  partial  revolutions  of  the 
graduated  fine  adjustment  the  working  distance  will  be  known.  For 
example,  suppose  it  required  5.5  revolutions  of  the  micrometer  screw 
to  raise  the  objective  from  the  surface  of  the  slide  where  the  object 
is  located  to  a  point  where  the  microscope  is  in  focus,  and  the  mi- 
crometer screw  raises  the  objective  o.i  mm.  for  each  complete  revolu- 
tion, then  the  total  elevation  will  be  o.i  x  5.5=  0.55  mm.,  that  is,  the 
working  distance  in  this  case  is  0.55  millimeter. 

§  79.  Free  working  distance  in  covered  objects.  —  Use  a  4  mm. 
objective  and  the  fly's  wing  or  any  covered  object.  Set  the  fine  ad- 


42  WORKING  DISTANCE  WITH  THE  MICROSCOPE       [Cn.  II 

justment  head  at  zero  (o).  Lower  the  objective  carefully  with  the 
coarse  adjustment  until  the  objective  just  touches  the  cover-glass. 
Now  focus  up  with  the  fine  adjustment  until  the  object  is  in  sharp 
focus,  noting  the  total  and  partial  revolutions  of  the  screw  to  accom- 
plish this.  The  distance  the  objective  was  raised  is  the  free  space 
between  the  front  of  the  objective  and  the  cover-glass.  Suppose  it 
required  3.2  revolutions  of  the  fine  adjustment  to  focus  the  objective, 
then  if  each  revolution  represents  o.i  mm.  the  total  elevation  is  3.2  x 
0.1=  0.32  mm.  for  the  free  working  distance  in  this  case. 

§  80.  Effect  of  the  cover-glass  on  the  working  distance.  —  It  is 
obvious  that  if  an  object  is  covered  with  a  layer  of  glass  that  the  free 
space  between  the  front  of  the  objective  and  the  object  will  be  lessened, 
and  if  the  layer  of  glass  is  considerably  thicker  than  the  working  dis- 
tance of  the  objective,  then  it  will  be  impossible  to  get  the  object  in 
focus.  If  the  layer  of  glass  is  relatively  thin,  then  it  will  be  possible 
to  focus  the  microscope  on  the  object,  but  from  the  law  of  refraction 
it  necessarily  follows  that  the  focus  of  the  microscope  with  and  without 
a  cover-glass  will  not  be  the  same. 

Now  from  the  refraction  of  the  rays  in  passing  from  one  medium  to 
another  of  different  refractive  power,  it  follows  that,  when  an  object 
is  in  or  below  a  stratum  of  glass  or  water  or  other  highly  refractive 
body,  the  object  will  appear  as  if  raised  (fig.  31,  51),  the  amount  of 
the  apparent  elevation  depending  on  the  refractive  index  of  the  cover- 
ing body,  —  the  greater  its  refraction,  the  more  the  apparent  elevation. 
The  general  physical  law  is  that  the  eye  being  in  the  air  the  apparent 
depth  of  an  object  below  the  surface  when  viewed  perpendicularly  is 
the  actual  depth  multiplied  by  the  reciprocal  of  the  index  of  refraction 
of  the  covering  body.  The  index  of  refraction  of  the  cover-glass  is 
1.52  or  approximately  1.50,  and  its  reciprocal  is  —^  =  f .  That  is, 
the  apparent  depth  is  only  f  its  actual  depth,  or  in  other  words  the 
object  seems  to  be  elevated  ^  of  the  actual  depth. 

Now  if  the  object  is  apparently  higher  up,  the  microscope  must 
be  raised  an  amount  equal  to  the  apparent  elevation  of  the  object. 
This  is  illustrated  in  fig.  31-32.  From  this  it  follows  that  the  free 
working  distance  of  the  objective  on  a  covered  object  is  not  lessened 
the  full  thickness  of  the  cover-glass,  but  only  f  of  that  thickness. 


CH.  II]     DETERMINING  THE  THICKNESS  OF  THE  COVER  43 

§  81.  Demonstration  that  the  working  distance  is  lessened  f 
the  thickness  of  the  cover-glass.  —  Use  a  clean,  flat  glass  slide.  Put 
an  ink  or  pencil  mark  on  the  upper  face  for  object.  Employ  a  16  mm. 
objective  and  8x  or  icx  ocular.  Focus  the  microscope  on  the  ink  or 
pencil  mark,  then  measure  the  free  space  between  the  slide  and  the 
end  of  the  objective  with  the  wooden  wedge,  as  directed  in  §  78.  This 
is  the  free  working  distance  (§77)  without  a  cover-glass. 

Cut  a  glass  slide  up  into  two  or  three  pieces  for  cover-glasses.  Meas- 
ure the  thickness  of  one  of  the  pieces  with  the  cover-glass  measurer  or 
in  some  other  good  way.  Place  this  over  the  mark  on  the  slide  which 
was  in  focus.  If  now  one  looks  into  the  microscope  the  mark  will 
not  be  in  focus  with  the  glass  cover  over  it.  Focus  up  carefully  until 
the  mark  is  again  in  focus.  Measure  the  space  between  the  top  of  the 
cover-glass  and  the  objective  with  the  work  as  before.  This  will 
represent  the  free  working  distance  with  this  cover-glass. 

Subtract  the  free  working  distance  with  this  cover-glass  from  that 
with  no  cover-glass  and  the  difference  will  be  the  amount  the  free 
working  distance  has  been  lessened  by  the  addition  of  the  cover. 
This  amount  compared  with  the  thickness  of  the  cover-glass  will 
give  the  ratio  of  lessening  of  working  distance  by  the  addition  of  the 
cover-glass. 

In  an  actual  case  the  results  were  as  follows: 

Free  working  distance  without  cover 4.62  mm. 

"        with  cover 3.54  mm. 

Lessening  of  the  working  distance  by  the  cover-glass 1.08  mm. 

The  actual  thickness  of  the  cover-glass  was 1.62  mm. 

That  is,  the  lessening  of  the  free  working  distance  was  not  so  great 
as  the  thickness  of  the  cover  (1.62  mm.),  but  less;  viz.  1.08  mm.; 
that  is,  in  the  proportion  of  y^rf  =  f  of  the  actual  thickness  of  the 
cover-glass. 

§  82.  Determining  the  thickness  of  the  cover-glass  with  mounted 
objects.  —  From  what  has  been  learned  about  the  free  working  dis- 
tance with  covered  objects,  it  is  possible  to  determine  the  thickness 
of  the  cover-glass  over  an  object  if  the  object  is  in  contact  with  the 
cover.  If  it  is  below,  as  shown  in  fig.  32,  and  the  mounting  medium 
is  Canada  balsam  with  approximately  the  same  refractive  index  as 


44  DETERMINING  THE  THICKNESS  OF  THE  COVER     [Cn.  II 

glass,  then  it  is  possible  to  determine  how  great  is  the  combined 
thickness  of  the  cover-glass  and  layer  of  Canada  balsam  over  the 
object. 

Demonstrate  the  method  as  follows:  (i)  Where  the  object  is  in 
contact  with  the  lower  surface  of  the  cover-glass  (fig.  31).  Use  a 
4  mm.  objective  and  a  cover-glass  y1/^  mm.  thick.  Make  a  black 
ink  mark  on  one  side  of  the  cover  and  a  colored  ink  mark  directly 
opposite  on  the  other  side  of  the  cover,  or  use  glass  pencils  of  two  colors. 
Set  the  graduations  of  the  fine  adjustment  at  zero  (o).  Place  the 
marked  cover  on  a  glass  slide,  and  put  under  the  micrdscope.  Focus 
with  the  coarse  adjustment  on  the  mark  at  the  upper  surface  of  the 
cover.  Then  focus  down  with  the  fine  adjustment  until  the  mark 
on  the  lower  surface  appears  sharp.  For  verification,  focus  up  until 
the  upper  mark  is  again  sharp.  The  elevation  will  of  course  be  the 
same  as  the  lowering.  If  the  total  and  partial  revolutions  of  the 
fine  adjustment  screw  are  noted,  they  will  show  how  much  the  objec- 
tive was  lowered  to  get  the  lower  mark  in  focus.  In  the  case  here 
given  it  was  lowered  i  revolution.  Now  as  each  revolution  moves 
the  objective  up  or  down  o.i  mm.  the  objective  was  moved  down  o.i 
or  TW  of  a  millimeter.  As  this  represents  f  of  the  thickness  of  the 
cover  from  the  effect  of  refraction,  the  whole  thickness  must  be  o.io 
-r-  §=  0.15  mm.  For  a  cover  of  unknown  thickness  with  the  object 
in  contact  with  its  under  surface,  put  an  ink  mark  on  the  upper  sur- 
face of  the  cover  and  proceed  exactly  as  above,  focusing  successively 
on  the  object  and  on  the  ink  spot. 

(2)  Where  the  object  is  somewhere  below  the  cover-glass  (fig.  32). 
In  this  case  the  thickness  of  the  cover-glass  cannot  be  determined, 
but  one  can  determine  very  approximately  the  combined  thickness 
of  the  cover-glass  and  the  mounting  medium  over  the  object  as  fol- 
lows: Put  an  ink  or  glass  pencil  mark  on  the  upper  surface  of  the  cover- 
glass.  Focus  the  mark  with  the  coarse  adjustment  after  setting  the 
graduations  of  the  fine  adjustment  at  zero  (o).  Then  focus  down  with 
the  fine  adjustment  until  the  object  is  sharp.  Note  the  number  of 
revolutions  and  the  partial  revolution  of  the  fine  adjustment  drum. 
As  this  amount  represents  only  f  of  the  actual  thickness  of  the 
glass  and  mounting  medium  over  the  object,  divide  the  observed 


CH.  II] 


LIGHTING  WITH  THE  MICROSCOPE 


45 


amount  of  movement  by  f  and  the  quotient  will  represent  the  total 
thickness  over  the  object. 

For  example,  in  one  case  the  microscope  was  focused  on  the  ink  mark 
at  the  top  of  the  cover,  and  then  it  was  necessary  to  focus  down  i§ 
revolutions  of  the  fine  adjustment  screw  to  bring  the  object  in  focus. 
That  is,  it  was  necessary  to  focus  down  0.15  mm.  Now  as  this  repre- 
sents but  f  of  the  actual  thickness  of  the  cover-glass  and  mount- 
ing medium  over  the  object,  the  entire  thickness  was  0.15^-!  = 
0.225  mm-  Probably  in  this  case  the  cover-glass  was  0.15  mm.  thick 
and  the  object  was  in  the  mounting  medium  0.075  mm-  below  the 
cover. 

LIGHTING  WITH  DAYLIGHT 

§  83.  Unmodified  sunlight  should  not  be  employed  except  in  special 
cases  (§  125).  North  light  is  best  and  most  uniform.  When  the 
sky  is  covered  with  white  clouds,  the  light 
is  most  favorable.  To  avoid  the  shadows 
produced  by  the  hands  in  manipulating  the 
mirror,  etc.,  it  is  better  to  face  the  light;  but 
to  protect  the  eyes  and  to  shade  the  stage 
of  the  microscope  some  kind  of  screen  should 
be  used.  The  one  shown  in  fig.  33  is  cheap 
and  efficient.  If  one  dislikes  to  face  the 
window  or  lamp  it  is  better  to^sit  so  that  the 
light  will  come  from  the  left,  as  in  reading. 

It  is  of  the  greatest  importance  and  ad- 
vantage for  one  who  is  to  use  the  micro- 
scope for  serious  work  that  he  should  com- 
prehend and  appreciate  thoroughly  the 
various  methods  of  illumination,  and  the 
special  appearances  due  to  different  kinds 
of  illumination. 

§  84.     Reflected,  incident,  or  direct  light. 
-  By  this  is  meant  light  reflected  upon  the 

object  in  some  way  and  then  irregularly  reflected  from  the  object  to 
the  microscope.  By  this  kind  of  light  objects  are  ordinarily  seen  by 
the  unaided  eye  and  the  simple  microscope  (fig.  4-5) .  In  Histology, 


jo    cm 


FIG.  33.  SCREEN  FOR 
SHADING  THE  MICROSCOPE 
AND  THE  OBSERVER. 

It  is  composed  of  heavy 
paper  hung  over  a  bent 
wire,  which  in  turn  is  an- 
chored in  a  small  tin  dish 
filled  with  lead. 


LIGHTING  WITH  THE  MICROSCOPE 


[CH.H 


reflected  light  is  but  little  used;  but  in  the  study  of  opaque  objects, 
like  whole  insects,  etc.,  it  is  used  a  great  deal.  For  a  simple  micro- 
scope and  low  powers  of  the  compound  microscope,  ordinary  day- 
light that  naturally  falls  upon  the  object,  or  is  reflected  or  condensed 

upon  it  with  a  mirror  or  condensing 
lens,  answers  very  well  (fig.  21  A,  34). 
For  high  powers  and  for  special  pur- 
poses, special  illuminating  apparatus 
has  been  devised  (fig.  50). 

§  85.  Transmitted  light.  —  By  this 
is  meant  light  which  passes  through 
an  object  from  the  opposite  side  (fig. 
21  B,  35).  The  details  of  a  photo- 
graphic negative  are  in  many  cases 
only  seen  or  best  seen  by  transmitted 
light,  while  the  print  made  from  it  is 
best  seen  by  reflected  light  (fig.  21  A, 

34). 

Almost  all  objects  studied  in  ani- 
mal and  vegetable  Histology  are 
lighted  by  transmitted  light,  and 
they  are  in  some  way  rendered  trans- 
parent or  semi-transparent.  The 
light  traversing  and  serving  to  illu- 
minate the  object  in  working  with 
a  compound  microscope  is  usually 
reflected  from  a  plane  or  concave 
mirror,  or  from  a  mirror  to  a  con- 
denser, and  thence  transmitted  to 
the  object  from  below  (fig.  20,  41). 
§  86.  Axial  or  central  light.  —  By 

this  is  meant  light  reaching  the  object  in  such  a  way  that  it  is  sym- 
metrically arranged  around  the  optic  axis  of  the  microscope,  then  the 
object  will  be  equally  illuminated  from  all  sides.  If  bundles  of  paral- 
lel rays  are  reflected  upon  the  object  from  the  mirror,  they  must  be 
so  disposed  that  the  object  will  receive  an  equal  quantity  of  light 


FIG.  34.  LOW-POWER  OBJEC- 
TIVE SHOWING  WORKING  DIS- 
TANCE AND  REFLECTED  LIGHT. 

Axis  The  principal  optic  axis 
of  the  objective  extended. 

SI  The  glass  slide  on  which  the 
object  is  mounted. 

O     Object. 

c     Cover-glass  over  the  object. 

W  The  working  distance  be- 
tween the  cover  and  the  objective. 

Mirror  The  mirror  is  repre- 
sented as  above  the  stage  and  re- 
flecting parallel  beams  upon  the 
object. 

FC  Front  combination  of  the 
objective. 

BC  Back  combination  of  the 
objective;  it  is  composed  of  a 
plano-concave  of  flint  (F)  and  a 
double  convex  lens  of  crown  glass 


CH.H] 


DIAPHRAGMS  AND  LIGHTING 


47 


from  all  sides.     If  the  bundles  of  light  are  made  up  of  diverging  or 
of  converging  cones,  then  the  axes  of  the  cones  should  be  coincident 
with  or  parallel  with  and  symmetrically 
arranged  around  the  optic  axis  of  the 
microscope  (fig.  41-42). 

§  87.  Oblique  light.  —  By  this  is 
meant  light  which  reaches  the  object 
with  its  axial  beam  oblique  to  the  optic 
axis  of  the  microscope.  With  oblique 
light  the  object  cannot  be  illuminated 
equally  from  all  sides,  but  largely  from 
one  side,  and  consequently  the  light  is 
said  to  be  unsymmetrical. 

If  no  condenser  is  used,  oblique  light 
is  obtained  by  turning  the  mirror  so 
that  parallel  rays  strike  the  object  ob- 
liquely to  the  optic  axis  of  the  micro- 
scope (fig.  35  c)  or  the  axis  of  the 
converging  or  diverging  beam  from  the 
concave  mirror  strikes  the  optic  axis 
obliquely  (fig.  35). 

If  a  condenser  is  used,  oblique  illumi- 
nation is  produced  by  making  the  dia- 
phragm opening  eccentric,  or  most 
simply  by  putting  the  finger  or  other 
opaque  body  between  the  mirror  and 
the  condenser  to  cut  off  part  of  the  light 
(fig.  46, 67).  The  end  result  in  all  cases 
is  that  the  object  is  lighted  unsym- 
metrically. 

§  88.    Use  of  a  diaphragm.  —  A  dia- 
phragm is  an  opaque  disc  with  an  opening,  and  is  placed  somewhere 
between  the  object  and  the  source  of  light. 

At  the  present  time  an  iris  diaphragm  is  almost  universally  em- 
ployed. It,  like  the  iris  of  the  eye,  can  be  expanded  or  contracted, 
and  thus  gives  a  large  range  of  openings  to  meet  different  conditions. 


FIG.  35.  HIGH-POWER  IM- 
MERSION OBJECTIVE  WITH  DI- 
RECT AND  OBLIQUE  TRANS- 
MITTED LIGHT. 

Axis  The  principal  optic  axis. 

Mirror  This  reflects  the 
light  up  through  the  object. 

A  B     Direct  light. 

C     Oblique  light. 

Stage  The  microscope  stage 
in  section. 

O     The  object. 

/  Immersion  liquid  between 
the  objective  and  object. 

F  C  The  front  lens  of  the 
objective. 

M  C  The  middle  combina- 
tion. 

B  C     The  back  combination. 


48  ARTIFICIAL  ILLUMINATION  [Cn.  II 

The  object  of  a  diaphragm  is  to  cut  off  adventitious  light  and  to 
-vary  the  aperture  to  suit  the  object  and  the  objective. 

§  89.  Size  and  position  of  the  diaphragm  with  a  mirror  only.  — 
When  no  condenser  is  used  in  addition  to  the  mirror,  a  diaphragm 
opening  about  the  size  of  the  front  lens  of  the  objective  may  be 
employed.  Its  position  may  be  close  to  the  object,  in  which  case  it 
admits  the  greatest  aperture  of  light,  and  cuts  off  the  most  adventi- 
tious light;  in  this  position  it  lights  the  smallest  field,  however. 

If  the  diaphragm  is  far  enough  below  the  object  the  field  may  all 
be  lighted,  but  the  aperture  will  be  smaller  than  when  it  is  close  to 
the  object,  as  one  may  see  by  removing  the  ocular  and  looking  down 
the  tube  into  the  back  lens  of  a  16  mm.  or  8  mm.  objective.  On  the 
other  hand,  while  the  aperture  of  the  objective  may  be  filled  even  with 
a  small  diaphragm  opening  close  to  the  object,  the  field  of  view  (§47, 
fig.  65)  may  be  but  partly  lighted.  In  that  case  the  opening  must 
be  increased  until  the  entire  field  is  illuminated.  One  must  learn 
by  practice  how  to  get  the  best  effects. 

§  90.  Diaphragm  with  condenser.  —  The  diaphragm  with  a  con- 
denser serves  to  vary  the  aperture  of  the  cone  of  light  to  adapt  it 
to  the  objective,  and  to  the  object. 

If  the  opening  in  the  diaphragm  is  not  in  the  axis  of  the  condenser 
the  object  will  be  unsymmetrically  illuminated;  the  object  will  also 
be  unsymmetrically  illuminated  if  the  diaphragm  is  wide  open  but 
the  light  blocked  from  one  side  by  placing  an  opaque  body,  like  the 
finger,  between  the  mirror  and  the  diaphragm  (fig.  46,  67). 

The  diaphragm  is  below  the  condenser  in  many  forms,  but  between 
the  lenses  in  some  (fig.  39-42). 

ARTIFICIAL  ILLUMINATION 

§  91.  Artificial  light.  —  While  daylight  is  to  be  preferred  for  most 
microscopic  as  for  other  exacting  work,  it  is  not  always  possible  to 
work  by  daylight,  and  then  sometimes  one's  work  room  or  laboratory 
is  so  situated  that,  even  in  the  daytime,  artificial  light  must  be  em- 
ployed. For  some  purposes,  like  photo-micrography,  it  is  desirable 
to  have  a  very  uniform  light,  and  this  is  gained  most  readily  by  using 
some  form  of  artificial  light. 


CH.  II]  DAYLIGHT   GLASS  49 

All  forms  of  artificial  light  have  been  used  at  some  time  for  micro- 
scopic work.  For  photography  and  for  drawing  (Chs.  VI-VII)  the 
arc  light  has  been  found  most  satisfactory.  For  the  usual  observa- 
tional work  with  the  microscope  the  effort  has  been  made  for  a  long 
time  to  get  an  artificial  light  which  should  approximate  daylight  as 
closely  as  possible.  This  desire  for  artificial  daylight  is  natural,  as  the 
eye  has  been  created  or  developed  for  daylight,  and  any  form  of  light 
differing  in  a  marked  degree  from  daylight  does  not  give  standard 
color  values,  and  is  liable  to  cause  eye  fatigue  if  some  parts  of  the 
visible  spectrum  are  markedly  brighter  than  with  daylight.  In  all 
of  the  ordinary  forms  of  artificial  light,  the  relative  intensity  toward 
the  red  end  of  the  spectrum  is  very  much  greater  than  with  daylight 
(fig.  36) ;  hence  color  values  are  distorted,  and  with  most  people  the 
excessive  red  intensity  produces  a  glare  and  lack  of  contrast  which  is 
trying  to  the  eyes. 

§  92.  Artificial  daylight.  —  For  the  production  of  artificial  day- 
light it  is  obvious  from  the  curve  here  shown  that  there  are  two  pos- 
sible means:  (i)  The  selection  of  two  kinds  of  artificial  light  in  which 
the  lack  in  one  is  made  good  by  the  excess  in  another,  and  by  mixing 
these  in  the  right  proportions  the  resulting  light  will  have  the  same 
relative  intensity  in  different  parts  of  the  spectrum  as  is  found  in  sun- 
light. This  is  the  "addative"  method  and  has  been  quite  success- 
fully realized  by  combining  a  mercury  arc  light  with  its  deficiency  in 
the  red,  but  its  richness  in  intensity  in  the  blue  end  of  the  spectrum, 
with  a  mazda  incandescent  lamp  with  its  excessive  red  intensity. 
If  these  two  lights  are  enclosed  in  a  glass  globe,  and  the  right  amount 
of  each  used,  very  good  daylight  is  produced. 

(2)  As  there  is  excessive  intensity  in  the  red  part  of  the  spectrum 
it  is  evident  that  if  this  excess  can  be  absorbed  by  a  light  filter  of  some 
kind,  then  also  the  relative  intensity  of  the  light  will  be  like  that  of 
natural  daylight.  This  is  the  "  sub  tractive "  method,  and  is  the 
method  employed  wherever  a  light  filter  or  colored  liquid,  colored 
gelatin,  colored  glass,  or  a  combination  is  used.  From  time  im- 
memorial various  colored  liquids  like  solutions  of  copper  salts  and 
colored  glasses  have  been  used  to  whiten  the  artificial  light. 

During  the  last  few  years,  however,  the  problem  has  been  solved, 


DAYLIGHT   GLASS 


[Cn.  II 


and  now  colored  glass  is  made  which  gives  to  artificial  light  true 
daylight  qualities.    As  each  artificial  light  has  its  own  special  curve 


12  Viol*        Blu« 


10 


I 


/ 


~\ — r 

Grw 


7 


~~1 — 1 — T 

•How  Or.ng. 


17   CD 


.41    .43    .45    .47    .49    .51     .53    .55    .57    .59    .61    .63    .65    .67    .69 
Wave  Length  in  Microns    M 

FIG.  36.     CURVE  OF  ENERGY  DISTRIBUTION  IN  MAZDA  C  LAMP,  THE  SUN  AND 
THE  LAMPLIGHT  FILTERED  THROUGH  DAYLIGHT  GLASS  (172  CD). 

(From  the  Sibley  Journal  and  the  Anatomical  Record). 

of  intensity  for  the  different  parts  of  the  spectrum,  naturally  a  special 
light  filter  must  be  worked  out  for  each  light  source.  Up  to  the 
present,  glass  filters  have  been  produced  for  the  welsbach  gas  light, 


CH.  II] 


DAYLIGHT    GLASS 


and  for  the  incandescent,  nitrogen-filled  tungsten  (mazda)  lamp. 
It  may  be  said  in  passing  that  these  glass  filters  whiten  any  artificial 
light,  but  that  true  daylight  color  values  are  given  only  under  the 
precise  conditions  for  which  the  glass  was  worked  out.  It  is  also 
gratifying  to  note  that  this  successful  solution  of  a  long  vexing 
problem  came  only  when  the  rigid  training  in  physics  and  chemistry 
and  the  facilities  of  a  great  manufacturing  plant  were  brought 
together. 

In  the  practical  use  of  these  daylight  filters  it  was  found  by  me  that 
the  surface  should  be  finely  ground  (frosted),  or  white  frosted  glass 
should  be  used  with  it.  Then  the  light  should  be  enclosed  in  some 
form  of  lantern  to  cut  off  all  unfiltered  light,  and  the  daylight  glass 


r 


I  v  1 

FIG.  37.    CHALET  MICROSCOPE  LAMP  IN  SECTION. 
(About  one  sixth  natural  size.) 

dg  —  dg  Windows  of  daylight  glass  about  82  mm.  square.  One  or  both  faces 
are  ground  with  very  fine  emery  or  carborundum  to  diffuse  the  light. 

me  Mazda  C  lamp  bulb  of  100  watts.  The  filament  of  the  lamp  is  opposite  the 
center  of  the  daylight  glass. 

S    The  lamp  socket  with  snap  switch  at  the  left. 

v,v,v  The  ventilating  flues.  The  one  at  the  bottom  goes  all  around;  the  ones  at 
the  top  are  under  the  roof  on  the  two  sides,  but  not  at  the  ends  (fig.  38).  The 
light  for  the  microscope  extends  directly  through  the  daylight  glass.  That  from 
the  windows  and  that  escaping  through  the  ventilating  flues  at  the  top  is  sufficient 
to  take  notes  by,  to  make  drawings  and  to  read. 

This  has  been  named  the  Chalet  Microscope  Lamp,  because  the  overhanging 
roof  for  shading  the  eyes  of  the  observer  gives  it  the  general  appearance  of  a  Swiss 
Chalet  (figs.  38, 58, 125). 


52  DAYLIGHT    GLASS  [Cn.  II 

placed  opposite  the  filament  of  the  lamp  as  shown  in  fig.  37,  where 
m  c  represents  the  filament. 

So  used,  the  brightly  illuminated  frosted  daylight  glass  becomes 
practically  the  source  of  light  for  the  microscope,  and  resembles  very 
closely  that  from  a  white  cloud.  There  is  no  glare,  the  color  values 
are  correct,  and  if  a  loo-watt,  nitrogen-filled,  mazda  lamp  is  used,  the 
light  is  abundant  for  all  powers  of  the  microscope  up  to  and  in- 
cluding the  1.5  mm.  oil  immersion. 

For  objectives  up  to  8  mm.  it  is  best  to  have  the  daylight  glass 
ground  on  both  sides.  The  diffusion  will  then  be  sufficient  to 
give  a  uniformly  lighted  field.  For  powers  of  4,  3,  2,  and  1.5 
mm,  focus  it  is  better  to  have  one  side  of  the  daylight  glass 
polished  and  one  side  ground.  This  gives  sufficient  diffusion  of 
the  light  from  the  source  to  fully  and  evenly  light  the  field,  and 
as  the  diffusion  is  less  the  brilliancy  of  the  light  will  be  corre- 
spondingly greater  from  the  smaller  area.  A  good  plan  is  to  have 
one  opening  of  the  lantern  (fig.  37-38)  with  a  disc  of  glass  ground 
on  both  sides  for  low  power  work  and  another  with  the  daylight 
glass  frosted  on  one  side  and  polished  on  the  other  for  high  powers. 

It  -may  be  said  that  the  position  of  the  ground-glass  filter  should 
be  close  to  the  source  of  light.  Its  brilliancy  will  vary  inversely  with 
the  square  of  its  distance  from  the  lamp  filament.  If  the  daylight 
glass  were  polished,  then  it  could  be  used  anywhere  between  the  source 
of  light  and  the  eye;  for  example,  under  the  condenser  in  the  usual 
place  for  polished  colored  glasses,  or  over  the  ocular.  The  advantage 
of  having  it  ground  and  near  the  lamp  filament  is  that  one  can  get  a 
uniform  light  and  wholly  avoid  the  image  of  the  lamp  filament.  Fur- 
thermore, when  using  a  lamp  it  should  be  enclosed  to  avoid  the  general 
flooding  of  the  room  with  unfiltered  light,  to  say  nothing  of  the  annoy- 
ance to  the  observer  and  his  coworkers.  The  enclosure  in  a  lantern 
(fig.  37-38)  avoids  all  that 


§  92a.  For  a  discussion  of  the  requirements  for  the  production  of  artificial 
daylight,  and  the  means  so  far  employed,  and  the  uses  of  artificial  daylight,  see: 

Herbert  E.  Ives.  Artificial  Daylight.  Journal  of  the  Franklin  Institute, 
vol.  177,  May,  1914,  pp.  471-499.  19  figures. 

Simon  H.  Gage.  Artificial  Daylight  for  the  Microscope.  Science,  N.  S.f 
vol.  42,  October,  1915,  pp.  534-536.  One  curve. 


CH.  II]         LIGHTING  WITH  DAYLIGHT  AND  A  MIRROR  53 

M.  Luckiesh.  Artificial  Daylight.  Science,  N.  S.,  vol.  42,  November,  1915, 
pp.  764-765- 

Henry  Phelps  Gage.  "  Daylite  Glass,"  a  color  screen  for  producing  day- 
light artificially.  The  Sibley  Journal  of  Engineering,  Ithaca,  N.  Y.,  Vol.  XXX, 
No.  8,  May,  1916.  4  quarto  pages,  6  figures. 

Simon  H.  Gage  and  Benjamin  F.  Kingsbury.  Some  apparatus  for  the 
microscopical  laboratory.  Anatomical  Record,  Vol.  X,  No.  8,  June,  1916, 
pp.  527-536.  7  figures  showing  the  use  of  the  daylight  glass  for  microscopic 
work. 

Anthony  J.  Brown.  Some  uses  of  artificial  daylight  in  the  psychological 
laboratory.  American  Journal  of  Psychology.  July,  1916,  Vol.  XXVII, 
pp.  427-429- 

LIGHTING  EXPERIMENTS  WITH  THE  SIMPLE  MICROSCOPE 

§  93.  Opaque  objects.  —  For  these  the  light  strikes  the  surface  and 
is  reflected,  mostly  in  an  irregular  manner  so  that  the  object  can  be 
seen  almost  equally  well  illuminated  from  any  angle.  Ordinarily 
the  daylight  falling  upon  the  object  will  sufficiently  illuminate  it,  also 
the  light  of  a  lamp. 

Place  a  printed  page  in  bright  daylight  or  near  a  lamp  where  the 
light  can  shine  upon  it  and  then  look  at  it  with  the  simple  microscope 
held  in  the  hand,  on  the  legs  of  the  tripod  (fig.  4,  17-19)  or  held  by  a 
special  stand.  By  varying  the  distance  between  the  microscope  and 
the  object  one  can  soon  find  the  best  focus,  and  by  changing  the 
position  of  the  object,  the  best  position  for  the  light  available. 

Of  course  if  one  wishes  to  discriminate  colors  precisely,  daylight, 
natural  or  artificial,  must  be  available. 

Take  some  object  in  the  hand  and  hold  it  in  a  good  light  and  then 
look  at  it  through  a  simple  microscope  held  in  the  other  hand. 

Remember  in  using  the  simple  microscope  that  the  eye  should  be 
near  the  microscope  to  see  the  largest  field  (§  15,  47,  57),  and,  as  will 
be  more  fully  shown  when  dealing  with  magnification,  the  nearer  the 
object  is  to  the  principal  focus  the  greater  will  be  the  apparent  increase 
in  size  (fig.  13-16). 

LIGHTING  EXPERIMENTS  WITH  THE  COMPOUND  MICROSCOPE 

§  94.    Daylight  with  a  mirror.  —  As  the  following  experiments  are 

for  mirror  lighting  only,  remove  the  substage  condenser  if  one  is  present 

(see  §  100,  for  condenser).     Place  a  mounted  fly's  wing  under  the 

microscope,  put  the  16  mm.  or  other  low  objective  in  position,  also 


54  ARTIFICIAL  LIGHT  FOR  THE  MICROSCOPE          [Cn.  II 

a  4x  or  5x  ocular.  With  the  coarse  adjustment  lower  the  tube  of  the 
microscope  to  within  about  i  cm.  of  the  object.  Use  an  opening  in 
the  diaphragm  about  as  large  as  the  front  lens  of  the  objective;  then 
with  the  plane  mirror  try  to  reflect  light  up  through  the  diaphragm 
upon  the  object.  One  can  tell  when  the  field  (§  47)  is  illuminated 
by  looking  at  the  object  on  the  stage,  but  more  satisfactorily  by  look- 
ing into  the  microscope.  It  sometimes  requires  considerable  manipu- 
lation to  light  the  field  well.  After  using  the  plane  side  of  the  mirror 
turn  the  concave  side  into  position  and  light  the  field  with  it.  As 
the  concave  mirror  condenses  the  light,  the  field  will  look  brighter 
with  it  than  with  the  plane  mirror.  It  is  especially  desirable  to  re- 
member that  the  excellence  of  lighting  depends  in  part  on  the  position 
of  the  diaphragm  (§  88).  If  the  greatest  illumination  is  to  be  obtained 
from  the  concave  mirror,  its  position  must  be  such  that  its  focus  will 
be  at  the  level  of  the  object.  This  distance  can  be  very  easily  deter- 
mined by  finding  the  focal  point  of  the  mirror  in  full  sunlight. 

§  96.  Use  of  the  plane  and  of  the  concave  mirror.  —  The  mirror 
should  be  freely  movable,  and  have  a  plane  and  a  concave  face  (fig. 
20).  The  concave  face  is  used  when  a  large  amount  of  light  is  needed, 
the  plane  face  when  a  moderate  amount  is  needed  or  when  it  is  neces- 
sary to  have  parallel  rays  or  to  know  the  direction  of  the  rays. 

EXPERIMENTS  WITH  ARTIFICIAL  LIGHT  AND  A  MIRROR 
§  96.  Lighting  with  a  kerosene  lamp.  —  For  this  a  lamp  with  a 
flat  wick  from  3  to  5  cm.  wide  is  best.  It  should  be  turned  up  well, 
but  not  enough  to  smoke.  The  face  of  the  flame  should  be  turned 
toward  the  microscope  for  low  powers.  For  moderate  powers  the 
flame  should  be  made  oblique  and  for  high  powers  the  edge  of  the  flame 
should  be  used.  This  is  because  the  thicker  source  of  light  gives  a 
greater  brilliancy.  Use  the  fly's  wing  or  any  well-stained  preparation. 
As  the  light  is  in  diverging  beams  it  is  best  to  use  the  concave  mirror 
to  partly  overcome  the  divergence.  One  must  learn  by  experience 
and  trial  how  far  off  to  have  the  lamp.  A  distance  of  15  to  20  cm. 
is  usually  satisfactory.  There  should  be  an  opaque  screen  between 
the  lamp  and  the  microscope  to  protect  the  eyes  of  the  observer  and 
to  screen  the  stage  of  the  microscope  (fig.  33). 


CH.  IlJ          ARTIFICIAL    LIGHT    FOR    THE    MICROSCOPE  55 

This  lamp  illumination  is  brilliant,  but  the  color  values  are  quite 
unlike  those  given  by  daylight. 

§  97.  Lighting  with  artificial  daylight.  —  For  the  source  of  light 
use  preferably  a  75-or  loo-watt  nitrogen-filled  mazda  lamp  enclosed 


FIG.  38.    LABORATORY  TABLE,  STOOL,  MICROSCOPE  AND  CHALET  LAMP  WITH 

DAYLIGHT  GLASS. 

(About  one  fifteenth  natural  size.) 

CL  Chalet  microscope  lamp  with  two  windows  of  daylight  glass  on  opposite 
sides  under  the  overhanging  roof.  The  roof  serves  to  protect  the  eyes  (fig.  37). 

M    Laboratory  microscope,  slightly  inclined. 

It  will  be  noted  that  the  table  rail  is  cut  out  in  front  to  avoid  interference  with 
the  knees  of  the  observer.  A  table  drawer  at  the  right  can  be  pulled  out  without 
moving.  The  revolving  piano  stool  can  be  adjusted  to  any  desired  height. 

in  a  kind  of  lantern  (figs.  37-38).  Have  the  lamp  filament  at  about 
the  level  of  the  center  of  the  microscope  mirror,  and  a  frosted  disc 
of  daylight  glass,  before  an  aperture  in  the  lantern.  The  aperture 
for  the  daylight  glass  should  be  from  5  to  10  cm.  in  diameter.  For 
all  high  powers  the  small  size  is  sufficient.  For  objectives  of  50  to 


56  ARTIFICIAL  LIGHT  FOR  THE  MICROSCOPE  [CH.  II 

100  mm.  equivalent  focus,  the  entire  field  might  not  be  lighted  with 
so  small  a  disc  of  daylight  glass. 

For  object,  use  a  fly's  wing  or  any  good,  well-stained  specimen. 
It  would  be  interesting  to  sit  near  a  window,  and  to  turn  the  mirror 
in  such  a  way  as  to  bring  in  daylight  a  part  of  the  time.  In  this  way 
one  can  get  a  good  idea  of  the  real  similarity  of  the  artificial  and  of 
the  natural  daylight.  If  one  also  had  an  electric  lamp  without 
any  light  filter  one  could  pass  in  order  from  real  daylight,  through 
the  artificial  daylight  and  then  on  to  the  unmodified  artificial  light. 
Without  seeing  these  in  comparison,  one  is  hardly  able  to  appreciate 
the  likeness  between  the  natural  and  artificial  daylight  and  the  great 
unlikeness  of  unfiltered  electric  light  and  artificial  daylight. 

CENTRAL  AND  OBLIQUE  LIGHT  WITH  A  MIRROR 
§  98.  Axial  or  central  light  (§86).  — Remove  the  condenser  or 
any  diaphragm  from  the  substage,  then  place  a  preparation  containing 
minute  air  bubbles  under  the  microscope.  The  preparation  may  be 
easily  made  by  beating  a  drop  of  mucilage  on  the  slide  and  covering 
it  (see  Chs.  IX-X).  Use  a  4  mm.  objective  and  a  4x  or  5x  ocular. 
Focus  the  microscope  and  select  a  very  small  bubble,  one  whose 
image  appears  about  i  mm.  in  diameter,  then  arrange  the  plane 
mirror  so  that  the  light  spot  in  the  bubble  appears  exactly  in  the 
center.  Without  changing  the  position  of  the  mirror  in  the  least, 
replace  the  air  bubble  preparation  by  one  of  Pleurosigma  angulatum 
or  some  other  finely  marked  diatom.  Study  the  appearance  very 
carefully. 

§  99.  Oblique  light  (§87).  —  Swing  the  mirror  far  to  one  side 
so  that  the  rays  reaching  the  object  may  be  very  oblique  to  the  optic 
axis  of  the  microscope.  Study  carefully  the  appearance  of  the  diatom 
with  the  oblique  light.  Compare  the  appearance  with  that  where 
central  light  is  used.  The  effect  of  oblique  light  is  not  so  striking  with 
histological  preparations  as  with  diatoms. 

It  should  be  especially  noted  in  §  98-99,  that  one  cannot  determine 
the  exact  direction  of  the  rays  by  the  position  of  the  mirror.  This  is 
especially  true  for  axial  light  (§98).  To  be  certain  the  light  is  axial 
some  such  test  as  that  given  in  §  195  should  be  applied. 


CH.  II]          LIGHTING  WITH  A  SUBSTAGE  CONDENSER  57 

CONDENSERS  OR  ILLUMINATORS 

§  100.  Condensers.  —  These  are  lenses  or  lens  systems  for  the 
purpose  of  illuminating  with  transmitted  light  the  object  to  be  studied 
with  the  microscope  (§  looa). 

For  the  highest  kind  of  investigation  their  value  cannot  be  over- 
estimated. They  may  be  used  either  with  natural  or.  artificial  light, 
and  should  be  of  sufficient  numerical  aperture  (N.A.)  to  satisfy  the 
widest  angle  objectives  to  be  used. 

It  is  of  great  advantage  to  have  the  substage  condenser  mounted 
so  that  it  may  be  moved  up  and  down  under  the  stage.  An  iris 
diaphragm  is  now  almost  universally  employed,  and  with  some  there 
is  a  scale  showing  the  numerical  aperture  (N.A.)  of  the  cone  of  light 
given  in  each  position  of  the  iris.  Finally  it  is  an  advantage  to  have 
a  stop  holder  and  diaphragms  with  central  stops  under  the  condenser 
for  the  production  of  dark-ground  illumination  (§  122). 

Condensers  or  illuminators  fall  into  two  great  groups,  the  achro- 
matic, giving  a  large  aplanatic  cone,  and  non-achromatic,  giving 
much  light,  but  a  relatively  small  aplanatic  cone  of  light. 

§  lOOa.  No  one  has  stated  more  clearly,  or  appreciated  more  truly  the 
value  of  correct  illumination  and  the  methods  of  obtaining  it  than  Sir  David 
Brewster,  1820,  1831.  He  says  of  illumination  in  general:  "The  art  of  illu- 
minating microscopic  objects  is  not  of  less  importance  than  that  of  preparing 
them  for  observation."  "The  eye  should  be  protected  from  all  extraneous 
light,  and  should  not  receive  any  of  the  light  which  proceeds  from  the  illuminat- 
ing center,  excepting  that  portion  of  it  which  is  transmitted  through  or  reflected 
from  the  object."  So  likewise  the  value  and  character  of  the  substage  con- 
denser was  thoroughly  understood  and  pointed  out  by  him  as  follows:  "I 
have  no  hesitation  in  saying  that  the  apparatus  for  illumination  requires  to 
be  as  perfect  as  the  apparatus  for  vision,  and  on  this  account  I  would  recom- 
mend that  the  illuminating  lens  should  be  perfectly  free  of  chromatic  and 
spherical  aberration,  and  the  greatest  care  be  taken  to  exclude  all  extraneous 
light  both  from  the  object  and  from  the  eye  of  the  observer."  See  Sir  David 
Brewster's  treatise  on  the  Microscope,  1837,  pp.  136,  138,  146,  and  the  Edin- 
burgh Journal  of  Science,  new  series,  No.  n  (1831)  p.  83. 

§  101.  Achromatic  condenser.  —  It  is  still  believed  by  all  expert 
microscopists  that  the  contention  of  Brewster  was  right,  and  the 
condenser  to  give  the  greatest  aid  in  elucidating  microscopic  structure 
must  approach  in  excellence  the  best  objectives.  That  is,  it  should 
be  as  free  as  possible  from  spherical  and  chromatic  aberration,  and 


LIGHTING  WITH  A  SUBSTAGE  CONDENSER          [Cn.  II 


therefore  would  transmit  to  the  object  a  very  large  aplanatic  cone  of 
light.  Such  condensers  are  especially  recommended  for  "photo-microg- 
raphy by  all,  and  those  who  believe  in  getting  the  best  possible  image 
in  every  case  are  equally  emphatic  that  achromatic  condensers  should 
be  used  for  all  work.  Unfortunately  good  condensers  like  good  ob- 
jectives are  expensive,  and  student  microscopes  as  well  as  many  others 
are  usually  supplied  with  the  non-achromatic 
condensers  or  with  none. 

Many  excellent  achromatic  condensers  have 
been  made,  but  the  most  perfect  of  all  seems  to 
be  the  apochromatic  of  Powell  and  Lealand  (Car- 
pen  ter-Dallinger,  p.  302).  To  attain  the  best 
that  was  possible  many  workers  have  adopted 
the  plan  of  using  objectives  as  condensers.  A 
special  substage  fitting  is  provided  with  the 
proper  screw  and  the  objective  is  put  into  posi- 
tion, the  front  lens  being  next  the  object.  As 
will  be  seen  below  (§  106-107),  the  full  aperture 
of  an  objective  can  rarely  be  used,  and  for  his- 
tological  preparations  perhaps  never,  so  that  an 
objective  of  greater  equivalent  focus,  i.e.,  lower 
power,  is  used  for  the  condenser  than  the  one  on 
the  microscope.  It  is  much  more  convenient, 
however,  to  have  a  special  condenser  with  iris 
diaphragm  or  special  diaphragms  so  that  one 
may  use  any  aperture  at  will,  and  thus  satisfy 
the  conditions  necessary  for  lighting  different  objects  for  the  same 
objective  and  for  lighting  objectives  of  different  apertures. 

§  102.  Non-achromatic  condensers.  —  Of  the  non-achromatic 
condensers  or  illuminators,  the  Abbe  condenser  or  illuminator  is  the 
one  most  generally  used.  From  its  cheapness  it  is  also  much  more 
commonly  used  than  the  achromatic  condenser.  It  consists  of  two  or 
three  very  large  lenses  and  transmits  a  cone  of  light  of  1.2  N.A.  to 
1.40  N.A.  (fig.  41,  46),  but  the  aberrations,  both  spherical  and  chro- 
matic, are  very  great  in  both  forms.  Indeed,  so  great  are  they  that 
in  the  best  form  with  three  lenses  and  an  illuminating  cone  of  1.40 


FIG.  39.  ACHROM- 
ATIC SUBSTAGE  CON- 
DENSER. 

(From  Watson's 
Catalogue) . 

1,2,3  The  lens  ele- 
ments making  up  the 
condenser.  While  the 
lenses  are  larger, 
the  general  construc- 
tion of  a  substage 
condenser  is  like  an 
objective,  i  represent- 
ing the  front,  w  the 
middle,  and  e  the  back 
combination  of  the 
objective. 


CH.  II] 


CENTERING  THE  SUBSTAGE  CONDENSER 


59 


N.A.,  the  aplanatic  cone  transmitted  is  only  0.5,  and  it  is  the  aplanatic 
cone  which  is  of  real  use  in  microscopic  illumination  where  details 
are  to  be  studied.  There  is  no  doubt,  however,  that  the  results  ob- 
tained with  a  non-achromatic  condenser  like  the  Abbe  are  much  more 
satisfactory  than  with  no  condenser.  The  highest  results  cannot 
be  attained  with  it,  however. 

§  103.  Position  of  the  condenser.  —  The  proper  position  of  the 
illuminator  for  high  objectives  is  one  in  which  the  beam  of  light  trav- 
ersing it  is  brought  to  a  focus  on 
the  object.  If  parallel  rays  are  re- 
flected from  the  plane  mirror  to  it, 
they  will  be  focused  only  a  few  milli- 
meters above  the  upper  lens  of  the 
condenser;  consequently  the  illumi- 
nator should  be  about  on  the  level 
of  the  top  of  the  stage  and  therefore 
almost  in  contact  with  the  lower  sur- 
face of  the  slide. 

§  104.  Determining  whether  the 
condenser  is  centered.  —  For  get- 
ting the  best  results  the  condenser 
should  be  centered  to  the  optic  axis 
of  the  microscope;  that  is,  the  optic  axis  of  the  condenser  and  of 
the  microscope  should  be  along  the  same  straight  line.  The  simplest 
method  of  determining  this  point  with  any  given  objective  is  to  get 
the  microscope  in  focus  on  some  very  small  or  transparent  object  and 
then  to  look  at  the  eye-point  above  the  ocular  with  a  magnifier.  While 
looking  at  the  eye-point  close  the  iris  diaphragm  until  the  aperture 
of  the  objective  is  only  partly  filled.  Now,  if  the  circle  of  light  appear- 
ing in  the  back  lens  is  in  the  center,  the  condenser  is  centered.  If 
it  is  not  in  the  middle,  then  the  condenser  is  not  centered  (fig.  43). 

If  the  condenser  has  centering  screws  (fig.  40),  it  is  very  simple  to 
adjust  them  until  the  circle  of  light  appears  in  the  center  of  the  back 
lens.  If  centering  screws  are  not  provided  and  the  condenser  is 
badly  decentered,  the  microscope  should  be  sent  to  the  makers  for 
correction. 


FIG.  40.  ACHROMATIC  SUBSTAGE 
CONDENSER  WITH  THE  DIAPHRAGM 
BETWEEN  THE  COMBINATIONS. 

(From  the  Catalogue  of  Zeiss). 
cs     Centering  screws. 


60  APERTURE  OF  THE  SUBSTAGE  CONDENSER         [Cn.  II 

§  105.  Testing  the  centering  by  slowly  opening  the  diaphragm.  - 
After  the  condenser  is  centered  as  well  as  possible  with  the  small  dia- 
phragm, one  can  test  its  centering  rigidly  by  looking  directly  down  the 
tube  without  the  ocular  or  by  looking  at  the  eye-point  with  a  magnifier 
and  slowly  opening  the  diaphragm.  If  it  is  accurately  centered,  the 
black  ring  will  become  narrower  and  disappear  all  around  at  the  same 
time.  If  it  is  not  accurately  centered  the  black  ring  will  open  on  one 
side.  In  case  the  condenser  is  not  found  centered,  change  its  position 
slightly  until  the  black  ring  disappears  at  the  same  time  all  around. 

§  106.  Numerical  aperture  of  the  condenser.  —  As  stated  above, 
the  aperture  of  the  condenser  should  have  a  range  to  meet  the  require- 
ments of  all  objectives  from  the  lowest  to  those  of  the  highest  aperture. 
It  is  found  in  practice  that  for  diatoms,  etc.,  the  best  images  are 
obtained  when  the  object  is  lighted  with  a  cone  which  fills  about  three- 
fourths  of  the  diameter  of  the  back  lens  of  the  objective  with  light, 
but  for  histological  and  other  preparations  of  lower  refractive  power 
only  one-half  or  one-third  the  aperture  often  gives  the  most  satis- 
factory images. 

To  determine  this  in  any  case  focus  upon  some  very  transparent 
object,  take  out  the  ocular,  look  down  the  tube  at  the  back  lens  or 
look  at  the  eye-point  (fig.  22-24)  with  a  magnifier.  If  less  than  three- 
fourths  of  the  back  lens  is  lighted,  increase  the  opening  in  the 
diaphragm  —  if  more  than  three-fourths,  diminish  it.  For  some 
objects  it  is  advantageous  to  use  less  than  three-fourths  of  the  aper- 
ture. Experience  will  teach  the  best  lighting  for  special  cases. 

§  107.  Aperture  and  source  of  light.  —  As  shown  by  Brewster 
nearly  a  century  ago,  and  as  brought  out  in  late  years  by  Gordon  and 
Wright,  the  amount  of  aperture  giving  the  best  results  depends  on  the 
size  of  the  source  of  light.  In  §  106  it  was  assumed  that  a  large  source 
was  used  like  a  window  or  door.  If  now  a  small  source  like  the  illu- 
minated disc  of  daylight  glass  in  the  lantern  for  artificial  daylight 
(fig.  37-38)  is  used,  then  considerably  more  of  the  aperture  of  the  ob- 
jective can  be  utilized  without  the  fogging  and  loss  of  detail  which 
results  when  using  a  large  source  like  a  window.  It  is  also  a  funda- 
mental fact  that  the  larger  the  aperture  utilized,  the  more  details,  and 
the  more  sharply  are  the  details  brought  out  in  the  specimen. 


CH.  II]     EXPERIMENTS  WITH  A  SUBSTAGE  CONDENSER  6 1 

Use  a  4  mm.  or  higher  objective  and  demonstrate  the  truth  of  the 
above  statements  by  looking  at  the  same  specimen,  using  the  window 
as  a  source  of  light  and  note  the  aperture  of  the  objective  which  gives 
the  best  effect.  Then  use  one  of  the  daylight  lanterns,  and  increase 
the  aperture  by  opening  the  diaphragm  until  the  best  image  is  obtained. 
Notice  the  detail  sharply  visible  in  the  two  cases. 

107a.  The  remarks  of  Wright,  Principles  of  Microscopy,  p.  219,  are  so 
pertinent  upon  this  point  that  they  are  here  repeated: 

"The  necessity  for  the  regulation  of  the  source  of  illumination  will  appear 
when  we  consider  the  optical  conditions  which  obtain  where  an  extended 
radiant  field  such  as  is  furnished  by  the  sky  or  a  broad  lamp  flame  is  employed 
as  a  source  of  light.  There  will  be  formed  in  such  a  case  upon  the  stage  of 
the  microscope  by  the  focused  condenser  an  image  of  the  light  source  which 
will  extend  beyond  the  limits  of  the  field  of  view  of  any  objective.  From  the 
radiant  points  included  within  this  illuminated  area  beams  will  pass  into  the 
aperture  of  the  objective.  Those  from  the  center  of  the  field  —  always  as- 
suming that  their  numerical  aperture  does  not  exceed  the  numerical  aperture 
of  the  objective  —  will  pass  through  the  aperture  unmutilated.  It  will  be 
different  with  respect  to  the  beams  which  proceed  from  the  periphery  of  the 
field.  These,  taking  the  aperture  obliquely,  will,  unless  in  the  case  where 
their  numerical  aperture  is  much  less  than  that  of  the  objective,  be  cut  down  in 
an  unsymmetrical  manner  by  the  margin  of  the  objective,  exactly  in  the  same 
way  as  would  be  the  case  if  transmitted  through  an  elliptical,  or,  in  the  extreme 
case,  through  a  slit  aperture. 

"  It  follows  that  while  the  radiant  points  in  the  center  of  the  field  will  be 
represented  in  the  image  by  circular  antipoints,  whose  dimensions  will  be 
determined  by  the  full  numerical  aperture  of  the  objective,  the  radiant  points 
on  the  periphery  of  the  field  will  be  represented  in  the  image  by  elliptical  or 
linear  antipoints  whose  long  axes  will  in  each  case  be  disposed  radially  to 
the  aperture,  overlapping  the  antipoints  in  the  center  of  the  field  in  such  a 
manner  as  to  fog  the  image." 

EXPERIMENTS  WITH  THE  CONDENSER  AND  ARTIFICIAL  LIGHT 

§  108.  Kerosene  lamp.  —  Use  a  kerosene  lamp  with  a  flat  wick. 
The  wick  should  be  from  3  to  5  cm.  wide.  For  use,  the  flame  is  turned 
up  well,  but  not  high  enough  to  smoke.  Put  the  lamp  15  to  20  cm. 
from  the  microscope.  Between  the  lamp  and  microscope  should  be 
a  dark  screen  sufficiently  high  and  broad  to  shade  the  microscope  stage 
and  to  protect  the  eyes  of  the  observer.  The  bottom  of  this  screen 
should  be  high  enough  from  the  table  to  admit  the  light  (fig.  33)  or 
preferably  it  should  have  a  hole  from  5  to  10  cm.  in  diameter  near  its 
lower  edge  to  admit  the  light  from  the  lamp  (fig.  58).  The  dark  screen 
with  the  hole  will  serve  also  to  hold  the  daylight  glass  (§  92). 


62  EXPERIMENTS  WITH  A  SUBSTAGE  CONDENSER      [Cn.  II 

The  greatest  brilliancy  comes  by  having  the  image  of  the  lamp 
flame  on  the  object  and  by  having  as  great  a  thickness  as  possible 
of  the  flame  toward  the  microscope. 

With  low  powers  the  face  of  the  flame  must  be  toward  the  micro- 
scope in  order  to  light  the  entire  field,  but  with  moderate  powers  it 
may  be  oblique,  and  with  the  highest  powers  the  edge  of  the  flame 
should  face  the  microscope,  as  this  utilizes  the  greatest  thickness  of 
the  flame.  Make  sure  that  the  flame  is  centered  by  moving  the  mirror 
or  the  lamp  (fig.  44). 

As  the  light  is  diverging  from  the  lamp  flame  it  is  usually  better 
to  employ  the  concave  mirror  in  order  to  overcome,  in  part,  this  diver- 
gence, and  light  a  larger  field.  In  some  cases  it  may  be  necessary 
also  to  lower  the  condenser  somewhat  to  get  the  entire  field  lighted. 
A  gas  lamp  with  an  incandescent  mantle  gives  good  results,  also  an 
acetylene  flame.  Proceed  in  general  as  with  the  kerosene  lamp. 

§  109.  Electric  lamp.  —  If  an  electric  bulb  is  used  it  should  be 
frosted  for  low  powers;  or  what  is  better,  a  clear  bulb  should  be  used 
and  a  piece  of  ground-glass  should  be  put  over  the  hole  in  the  screen 
between  the  lamp  and  the  microscope  (fig.  58).  The  lamp  should  be 
close  to  the  ground-glass. 

For  moderate  and  high  powers  the  ground-glass  is  excellent  also. 
For  lighting  the  entire  field  it  may  be  necessary  to  use  the  concave 
mirror  and  also  to  lower  the  condenser. 

For  the  highest  powers  a  single  filament  of  the  incandescent  lamp 
is  focused  on  the  object  and  carefully  centered.  For  lighting  the 
entire  field  lower  the  condenser  slightly  and  thus  spread  the  light 

(fig.  42). 

Both  mirrors  should  be  tried  alternately,  also  raising  and  lowering 
the  condenser  a  small  amount.  One  can  thus  find  which  arrangement 
gives  the  best  possible  results  in  difficult  cases.  While  it  may  take 
some  time  to  try  the  different  things,  in  the  long  run  it  will  pay  in  the 
satisfaction  given  by  the  more  perfect  image. 

For  the  electric  arc  with  the  microscope  see  under  drawing  and 
photography  (Chs.  VI-VII). 

§  110.  Aperture  and  diaphragm.  —  It  is  to  be  remarked  that  with 
a  very  small  source  of  light  the  entire  aperture  of  the  objective  may  be 


CH.  II]       EXPERIMENTS  WITH  A  SUBSTAGE  CONDENSER 


filled  if  a  proper  illuminator  or  condenser  is  used.  This  is  because 
light  rays  go  off  from  a  light  source  in  the  form  of  a  sphere,  and 
even  a  point  source  would  give  light  which  would  fill  any  aperture. 
The  aperture  in  a  given  case  depends  on  the  diaphragm  used  with  the 
condenser,  and  the  size  of  the  diaphragm  must  vary  directly  as  the 
aperture  of  the  objective.  That  is,  it  is  just  the 
reverse  of  the  rule  for  diaphragms  where  no  con- 
denser is  used  (§  89) ;  for  there  the  diaphragm  is 
made  large  for  low  powers,  and  consequently  low 
apertures,  while  with  the  condenser  the  diaphragm 
is  made  small  for  low  and  large  for  high  powers, 
as  the  aperture  is  greater  in  the  high  powers  of 
a  given  series  of  objectives.  It  is  very  instructive 
to  demonstrate  this  by  using  a  16  mm.  objective 
and  opening  the  diaphragm  of  the  condenser  till 
the  back  lens  is  just  filled  with  light.  Then  if  one 
uses  a  4  mm.  objective  it  will  be  seen  that  the 
back  lens  of  the  higher  objective  is  only  partly 
filled  with  light  and  to  fill  it  the  diaphragm  must 
be  much  more  widely  opened. 

§  111.  Mirror  and  light  for  the  condenser.  - 
It  is  best  to  use  light  with  parallel  rays.  The 
rays  of  daylight  are  practically  parallel;  it  is  best, 
therefore,  to  employ  the  plane  mirror  for  all  but 
the  lowest  powers.  If  low  powers  are  used  the  whole  field  is  not 
illuminated  with  the  plane  mirror  when  the  condenser  is  close  to  the 
object;  furthermore,  the  image  of  the  window  frame,  objects  out- 
side the  building,  as  trees,  etc.,  would  appear  with  unpleasant  dis- 
tinctness in  the  field  of  the  microscope.  To  overcome  these  defects 
one  can  lower  the  condenser  and  thus  light  the  object  with  a  diverg- 
ing cone  of  light,  or  use  the  concave  mirror  and  attain  the  same  end 
when  the  condenser  is  close  to  the  object  (fig.  42). 

§  112.  Lighting  the  entire  field  with  a  condenser.  —  With  the 
condenser  there  are  two  conditions  that  must  be  fulfilled:  the  proper 
aperture  must  be  used,  and  the  whole  field  must  be  lighted.  As  seen 
in  §  no  the  diaphragm  of  the  condenser  regulates  the  aperture  of  the 


FIG.  41.  SUBSTAGE 
CONDENSER  OF 
ABBE  WITH  PARAL- 
LEL LIGHT. 

Axis  The  prin- 
cipal optic  axis  of 
the  condenser  and 
objective  '(Ob). 

The  iris  dia- 
phragm (D)  is  be- 
low the  condenser. 


64 


EXPERIMENTS  WITH  A  SUBSTAGE  CONDENSER     [Cn.  II 


illuminating  cone,  but  has  nothing  to  do  with  lighting  a  large  or  a 
small  field.  The  size  of  field  that  is  lighted  by  means  of  a  condenser 
can  be  modified  in  two  ways: 

(i)   Suppose  that  the  image  of  the  source  of  light  is  focused  on  the 
object,  the  size  of  that  image  will  determine  the  size  of  field  which  is 
illuminated  in  a  given  case.     If  the  illuminated  field  is  not  so  large 
as  the  objective  field,  then  the  source  of  light  is 
too  small,  or  too  far  away.     In  that  case  use  a 
larger  source  or  bring  the  source  closer  to  the 
microscope. 

(2)  By  lowering  the  condenser  or  using  the  con- 
cave mirror  a  much  larger  object  can  be  fully 
lighted,  as  it  is  in  a  diverging  cone  of  light  above 
the  focal  point  of  the  condenser  where  the  light 
is  spread  over  a  greater  area  (fig.  42). 

For  quite  low  objectives,  35  to  60  mm.  focus,  it 
is  better  to  remove  the  condenser  and  use  the 
mirror  only.  The  whole  field  can  be  illuminated 
easily  and  sufficiently  in  this  way. 

§  113.  Homogeneous  immersion  condenser 
(Ch.  IX).  —  For  numerical  apertures  higher  than 
i. oo  it  is  necessary  to  connect  the  under  side  of 
the  microscopic  slide  with  the  top  of  the  con- 
denser with  homogeneous  immersion  fluid.  As 
the  objectives  used  for  high  apertures  are  also 
homogeneous  immersion,  there  is  no  change  in  the  direction  of  the 
light  from  the  condenser  until  it  reaches  the  objective,  excepting  dif- 
fraction effects,  and  the  reflecting  or  refracting  action  of  the  speci- 
men in  the  path  of  the  rays.  The  special  need  of  the  homogeneous 
connection  between  the  slide  and  the  condenser  comes  from  the  laws 
of  refraction.  If  there  were  not  a  homogeneous  connection  the  most 
oblique  rays,  that  is,  those  giving  a  numerical  aperture  above  i.oo 
with  a  solid  cone  of  light,  and  the  most  oblique  rays  with  a  hollow 
cone  of  light,  would  meet  the  terminal  surface  of  the  condenser  at  an 
angle  greater  than  the  critical  angle,  41°  +  ,  and  would  therefore  be 
totally  reflected  as  from  the  surface  of  a  mirror,  and  the  advantage 


FIG.  42.  SUB- 
STAGE  CONDENSER 
WITH  CONVERGING 
LIGHT. 

A  xis  The  prin- 
cipal optic  axis  of 
the  condenser  and 
the  objective  (Ob). 
The  iris  diaphragm 
(D)  is  below  the  con- 
denser. 


CH.  II]     EXPERIMENTS  WITH  A  SUBSTAGE  CONDENSER  65 

of  the  condenser  be  in  part  nullified  (see  Ch.  IX  for  critical  angle). 
By  making  the  homogeneous  connection  between  the  slide  and  con- 
denser, any  light  leaving  the  condenser  passes  on  through  the  slide 
without  change  of  direction,  except  any  that  may  be  given  by  the 
object. 

The  centering  of  the  homogeneous  immersion  condenser  is  of 
prime  importance,  and  practically  all  forms  of  them  are  supplied  with 
centering  screws  for  the  pur- 
pose (fig.  40,  §  104). 

§  114.     Use    of     artificial 
daylight.  —  Place  a  piece  of 

the    daylight    glass    in    the  FlG;43_44>     CENTERING  THE  CONDENSER 

opening  of   the   dark   screen  AND  THE  SOURCE  OF  LIGHT. 

or   lantern    (fig.    37-38).     If  A     The  spot  of  light  in  the  back  com- 

this  daylight  glass  is  lightly  bination  of  the  objective  at  one  side,  show- 

j      -±        -n     j-rc           4.1-  ing  that  the  condenser  is  not  centered, 
ground,    it    will    diffuse    the  B     The  spot  of  light  in  the  center,  show- 
light  without  lowering  its  in-  ing  that  the  condenser  is  centered. 

,,  C     The  source  of   light   is   not  in    the 

tensity  too  greatly.  middle 

If  possible,  use  a  IOO  D  The  source  of  light  is  centered  and  il- 
watt  nitrogen-filled  tungsten  luminate: 

(mazda)  concentrated  filament  lamp.  Put  the  lamp  within  5  to 
10  cm.  of  the  daylight  glass.  Use  low  and  high  powers  and  the 
various  colored  specimens  that  require  daylight  to  bring  out 
their  color  values.  No  matter  how  delicate  the  coloring  or  what 
the  tint  is,  it  will  be  as  faithfully  presented  by  the  artificial  day- 
light as  by  natural  daylight.  One  can  prove  this  by  having  the 
apparatus  near  a  window,  then  the  mirror  can  be  turned  toward 
the  artificial  or  the  natural  daylight  at  will  and  the  color  effects  can 
be  compared. 

By  using  the  daylight  glass  with  other  artificial  lights  it  will  be  seen 
that  even  those  lights  when  filtered  through  this  glass  give  remarkably 
good  daylight  effects. 

§  115.  Axial  and  oblique  light  with  the  condenser.  —  To  demon- 
strate the  effect  of  the  methods  of  illumination  when  a  condenser  is 
used,  take  any  striking  preparation  like  a  diatom  (Pleurosigma  angu- 
latum,  for  example);  employ  a  4  mm.  objective.  Being  sure  that 


66  EXPERIMENTS  WITH  A  SUBSTAGE  CONDENSER       [Cn.  II 

the  condenser  is  centered,  fill  the  aperture  of  the  objective  about 
3/4  full  of  light  (§  no).  Study  the  preparation  with  the  central 
light  and  note  the  appearance  of  the  markings.  Cover  a  part  of  the 
diaphragm  opening  by  putting  the  finger  or  some  other  opaque  object 
between  it  and  the  mirror  (fig.  46).  Note  that  the  markings  come  out 
more  strongly.  Hold  the  finger  in  position  and  open  the  diaphragm 
widely  and  see  if  the  markings  can  still  be  made  out.  Now  remove 
the  finger  so  that  the  object  is  lighted  by  the  full  aperture  of  central 
light.  Probably  the  markings  will  not  appear  at  all.  Put  the  finger 


Objectly*  7?  Bach  of  C         Objective 


FIG.  45.     APERTURE  OF  THE  SUBSTAGE  CONDENSER  AND  OF  THE  OBJECTIVE. 
(From  Nelson,  Jour.  Roy.  Micr.  Soc.). 

A     The  cone  of  light  from  the  condenser  fills  the  aperture  of  the  objective  (B) 
D     The  cone  of  light  of  the  condenser  only  partly  fills  the  aperture  of  the 

objective  (C). 

In  A  and  D  the  condenser  and  objective  are  shown  in  section;   in  B  and  C, 

the  back  lens  of  the  objective  is  shown  in  face  view  as  when  looking  down  upon 

it. with  the  ocular  removed. 

back  in  position  to  give  oblique  light  and  the  markings  will  again  be 
seen.  Remove  the  finger  and  slowly  close  up  the  diaphragm.  When 
the  proper  aperture  is  reached  the  markings  will  again  appear. 

For  histological  preparations  the  oblique  light  is  not  a  help  in  bring- 
ing out  details  of  structure.  There  the  end  is  reached  by  using  the 
proper  aperture,  regulating  the  source  of  light,  and  by  differential 
staining  (Chs.X-XI). 

§  116.  Lateral  swaying  of  the  image.  —  Frequently  in  studying 
an  object,  especially  with  a  high  power,  it  will  appear  to  sway  from 
side  to  side  in  focusing  up  or  down.  A  glass  stage  micrometer  or  fly's 
wing  is  an  excellent  object.  Make  the  light  central  or  axial  and  focus 
up  and  down  and  notice  that  the  lines  simply  disappear  or  grow  dim. 


CH.  II] 


DARK-GROUND  ILLUMINATION 


67 


Now  make  the  light  oblique,  either  by  making  the  diaphragm  open- 
ing eccentric  or,  if  simply  a  mirror  is  used,  by  swinging  the  mirror  side- 
wise.  On  focusing  up  and  down,  the  lines  will  sway  from  side  to  side. 
What  is  the  direction  of  apparent  movement  in  focusing  down  with 
reference  to  the  illuminating 
ray?  What  in  focusing  up?  If 
one  understands  the  experiment 
it  may  sometimes  save  a  great 
deal  of  confusion.  (See  under 
testing  the  microscope  for  sway- 
ing with  central  light,  §  166.) 

DARK-GROUND  ILLUMINATION 

§  117.  Dark-ground  illumi- 
nation. —  By  this  is  meant  that 
form  of  illumination  in  which 
the  object  appears  light  and  the 
background  dark.  The  appear- 
ance is  something  like  a  series 
of  bright  objects  in  a  dark  night. 

In  order  to  be  available  for 
dark-ground  illumination  ob- 
jects must  be  in  a  refracting 
medium  different  from  them- 
selves, and  must  have  either 
strongly  refracting  or  reflecting 
•qualities. 

The  optical  arrangements  for 
this  form  of  illumination  must 
be  such  that  the  object  is  lighted 

by  a  beam  of  light  which  cannot  get  into  the  objective  either  because 
the  rays  are  so  oblique,  or  because  they  are  cut  out  before  reaching 
the  eye.  In  either  case  the  light  on  the  background  never  reaches  the 
eye.  Only  that  which  is  reflected,  refracted,  or  diffracted  by  the 
object  reaches  the  eye.  Consequently  the  appearance  is  of  a  bright 
object  in  a  dark  field. 


FIG.  46. 


OBLIQUE  LIGHT  WITH  A 
CONDENSER. 

(From  Chamot). 


The  iris  diaphragm  is  opened  com- 
pletely and  the  light  from  one  side  is 
blocked  out  by  inserting  the  finger;  this 
gives  unsymmetrical  light  and  all  of  it  is 
oblique  to  the  optic  axis. 


68 


DARK-GROUND    ILLUMINATION 


[CH.  II 


§  118.  Dark-ground  illumination  by  reflected  light.  —  This  is  the 
simplest  method  of  getting  the  appearance  of  shining  or  white  objects 
in  a  dark  field.  Use  a  i6-mm.  or  lower  objective.  For  object  use  a 
glass  slide  with  particles  of  lint,  flour  or  starch,  or  other  powder  dusted 
on  the  slide.  Put  a  piece  of  black  velvet,  or  other  dull  black  surface, 
over  the  opening  in  the  stage  and  then  put  the  slide  in  position.  Place 
the  microscope  near  a  well-lighted  window  or  use  artificial  light  and 
let  it  shine  on  the  slide.  If  now  one  looks  into  the  microscope,  the 
particles  of  dust  on  the  slide  will  appear  bright  and  the  field  will  be 
dark.  In  the  subsequent  experiments  (§  119,  121)  remember  that 
the  appearance  may  be  due  to  light  falling  on  top  of  the  specimen, 
as  here,  and  not  that  passing  up  from  below.  The  top  light  can 
be  eliminated  by  shading  the  stage  with  the  hand  or  a  black  card. 

§  119.      Dark-ground      illumination 
with  light  from  below   the    stage.  — 
Swing  the  substage  aside,  use  a  i6-mm. 
or  lower  objective.     For  object  dust 
some   starch   grains   on  a  glass  slide. 
Swing  the  mirror  far  to  the  side  and 
Lam    W      direct  the  ^ght  very  obliquely  upon 
FIG.  46a.     DARK-FIELD  Iixum-    the  object.    If  the  light  is  sufficiently 
NATION  BY  LIGHT  FROM  BELOW  THE    oblique  the  grains  will  shine,  as  if  self- 

luminous  in  a  dark  field.     Use  central 
soIbX'S^ofUct'^r   Hght  and  the  grains  will  be  dark  on  a 

the  objective  directly,  and  the  ob-    white  field. 


deflected   rays   are   indicated   by    (fig.   46a)    Read's   original   experiment 

can  be  tried  with  striking  results. 


broken  lines. 


DARK-GROUND  ILLUMINATION  WITH  THE  CONDENSER 

§  120.  Put  the  condenser  back  in  place  and  close  up  under  the 
stage.  As  with  the  mirror  alone  the  light  striking  the  object  must 
be  so  oblique  to  the  axis  of  the  microscope  that  it  will  get  into  the 
objective  only  when  reflected  or  refracted  by  the  object.  This  may 
be  accomplished  in  two  ways. 

§  121.  By  lighting  from  one  side  as  with  the  mirror  only.  —  Use 
a  i6-mm.  or  lower  objective;  for  object,  a  glass  slide  with  dust  particles. 


CH.  II] 


DARK-GROUND    ILLUMINATION 


FIG.  47.  REFRACTING  OR 
BRIGHT-FIELD  CONDENSER 
WITH  CENTRAL  -  STOP  TO  GIVE 
DARK-FIELD  ILLUMINATION. 

The  condenser  is  shown  in 


Open  the  substage  diaphragm  to  its  full 
extent  and  reflect  the  light  up  through 
the  condenser.  The  particles  will  now 
appear  dark  on  a  white  field.  Make  the 
light  unsymmetrical  and  very  oblique 
by  putting  the  finger  in  the  path  of  the 
light  on  one  side  (fig.  46)  and  the  field  will 
be  dark  and  the  particles  bright. 

§  122.  By  lighting  with  a  hollow 
cone.  —  This  lights  the  object  with  a  ring 
of  light,  all  the  rays  of  which  are  so 
oblique  that  they  fall  outside  the  objec- 
tive (fig.  47).  Some  of  this  very  oblique 
light  is  deflected  by  the  object  into  the 
microscope  objective,  and  thence  passes 
to  the  eye.  The  question  naturally  arises 
how  one  is  to  determine  the  size  of  the 

central  Stop  to  use  with  a  given  con-  and  continuing  wholly  outside 
denser  and  objective.  This  is  easily 
determined  as  follows :  The  field  is  lighted 
well  as  for  ordinary  bright-field  observa- 
tion, and  the  microscope  is  then  focused 
on  some  object.  The  object  is  then 
removed  and  the  iris  diaphragm  of  the 
condenser  opened  to  its  fullest  extent. 
If  one  then  removes  the  ocular  and  looks 
down  the  tube  of  the  microscope  the  back 
lens  will  be  seen  to  be  fully  illuminated 
(fig.  456).  If  now  the  iris  diaphragm  is 
slowly  closed  it  will  be  seen  to  reach  the 
margin  of  the  back  lens,  and  if  closed  still 
farther  the  light  will  be  cut  out  around 
the  margin.  The,  size  of  the  central  stop 
to  use  is  the  opening  in  the  iris  diaphragm 
when  the  back  lens  is  just  filled  with  light.  This  opening  in  the 
iris  can  be  measured  by  dividers,  and  then  a  central  stop  prepared 


The  light  deflected  by  the  ob- 
ject into  the  objective  is  shown 
by  broken  lines. 

Axis  The  principal  optic 
axis  of  the  condenser  and  of 
the  microscope. 

CS  Central  stop  to  cut 
out  the  middle  rays  of  the  solid 
cone  of  light.  Above  it  is 
shown  in  section,  and  below  in 
face  view. 

Glass  Slide  The  slip  of  glass 

Tn  which  the  object  is  mount- 
It  is  advantageous  to  have 
the  slide  in  homogeneous  im- 
mersion contact  with  the  top  of 
the  condenser  (§  126). 

Object  The  object  to  be 
studied  by  dark-field  illumina- 
tion. 

Objective  The  front  lens  of 
the  microscope  objective. 


70  DARK-GROUND    ILLUMINATION  [Cn.  II 

from  bristol  board  like  that  in  visiting  cards.  It  is  well  to  blacken 
the  stop  with  India  ink.  If  such  a  stop  is  used  all  the  light 
which  would  enter  the  objective  directly  is  shut  out,  and  only 
that  which  is  deflected  by  the  object  will  get  into  the  micro- 
scope. If  the  above  test  is  made  with  different  objectives  it  will  be 
found  that  the  size  of  the  central  stop  varies  directly  with  the  aper- 
ture, that  is,  the  greater  the  aperture  the  larger  must  be  the  central 
stop;  and  conversely  the  larger  the  central  stop  the  narrower  will 
be  the  open  ring  allowing  the  light  to  pass  through  the  condenser  to 
form  the  hollow  cone  of  light.  It  follows  from  this  that  with  the 
higher  objectives  where  more  light  is  needed  the  ring  of  light  is  nar- 
rower; hence  a  stronger  light  must  be  used  for  a  high  than  for  a  low 
objective  or  the  objects  will  not  be  lighted  brilliantly  enough  so  that 
they  can  deflect  sufficient  light  into  the  microscope  to  make  them 
satisfactorily  visible. 

After  inserting  the  proper  central  stop,  put  a  slide  under  the  mi- 
croscope and  scatter  some  flour  or  starch  upon  it;  then  open  the 
iris  diaphragm  to  its  full  extent,  and  the  particles  of  flour  or  starch 
will  shine  as  if  they  were  self-luminous  (see  fig.  $of,  lower  half).  In 
case  the  field  looks  gray  instead  of  black,  either  the  central  stop  is 
not  large  enough  to  cut  out  all  the  light  from  the  condenser,  or  the- 
particles  of  flour  or  starch  are  too  numerous,  not  leaving  enough 
blank  space  between  them. 

§  123.  Dark-ground  illumination  by  refraction.  —  In  the  ex- 
periments already  given  for  dark-ground  effects  the  particles  of  dust 
have  reflected  the  very  oblique  light  into  the  microscope  objective. 
The  same  effect  may  be  produced  by  minute  bodies  refracting  the  very 
oblique  light  and  thus  turning  part  of  it  into  the  objective  of  the 
microscope.  There  are  two  cases: 

(i)  Objects  whose  refraction  is  less  than  the  mounting  medium: 
For  this  use  air  bubbles.  Make  a  preparation  by  beating  on  a  slide 
with  a  knife  blade  a  small  drop  of  gum  arabic  mucilage  or  other 
transparent  viscid  substance  like  saliva.  This  will  include  many 
air  bubbles.  Put  the  preparation  under  the  microscope,  using  the 
i6-mm.  or  lower  objective  as  before.  Try  an  8x  or  lox  ocular.  Use 
the  ring  diaphragm  and  light  as  well  as  possible.  The  air  bubbles 


CH.  II]    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    71 

will  shine  like  globules  of  silver  in  a  dark  field.  By  focusing  care- 
fully the  image  of  the  ring  diaphragm  will  be  seen  in  the  air  bubbles. 
The  spherical  air  bubbles  act  like  concave  lenses  in  the  surrounding 
medium  of  greater  refractive  power,  and  by  changing  the  direction 
of  the  rays  passing  through  them  turn  some  of  them  into  the  objec- 
tive, hence  the  appearance.  If  one  uses  saliva  as  liquid,  the  irregular 
gray  bodies  seen  are  epithelial  cells  from  the  mouth. 

(2)  Objects  having  a  greater  refractive  power  than  the  surround- 
ing medium:  Most  objects  studied  belong  to  this  group.  For  object 
use  milk  diluted  four  or  five  times  with  water  or  make  an  oil-globule 
preparation  by  beating  in  a  large  drop  of  water  a  small  drop  of  oil. 
,  The  oil  globules  are  more  refracting  than  the  surrounding  liquid, 
hence  will  act  as  convex  lenses.  But  the  difference  in  refraction  is 
not  so  great  as  with  air  and  water;  hence  the  dark  center  will  be 
wider  and  the  bright  ring  narrower.  One  can  also  see  the  image  of 
the  ring  diaphragm,  but  the  central  stop  is  relatively  larger  than  with 
the  air  bubbles. 

§  124.  Infusoria  with  dark-ground  illumination.  —  A  very  striking 
and  instructive  preparation  for  dark-ground  illumination  may  be 
made  by  taking  water  well  supplied  with  living  infusoria  and  other 
micro-organisms  as  object.  These,  under  the  microscope  when  prop- 
erly lighted  as  indicated  above  (§  123),  appear  like  shining  creatures 
swimming  in  black  ink  (§211). 

DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS 

§  125.  For  this  a  special  condenser  which  gives  a  very  oblique 
beam  is  required.  As  was  pointed  out  by  Wenham  1850-1856,  re- 
fracting condensers,  like  those  considered  above  (§  100-113),  are  not 
so  well  adapted  for  obtaining  a  suitable  hollow  cone  of  light  for  dark- 
field  work  as  a  reflecting  condenser.  This  is  due  to  the  difficulty 
in  getting  rid  of  the  spherical  and  chromatic  aberration  in  the  re- 
fracted rays  of  light  at  such  great  apertures.  In  the  best  reflecting 
condensers  the  aperture  of  the  rays  in  the  hollow  cone  of  light  ranges 
from  i. oo  N.A.  for  the  lower  limit  (fig.  48)  to  1.40  N.A.  for  the  upper 
limit.  The  rays  at  an  aperture  below  i.oo  N.A.  are  cut  out  by  a 
central  stop  (fig.  48). 


72     DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS   [Cn.  II 

§  125a.  Reducing  diaphragm  for  oil  immersion  objectives.  —  It  necessarily 
follows  that  if  the  condenser  transmits  light  between  the  apertures  of  i.oo  and 
1.40  N.A.,  that  the  objectives  used  must  have  an  aperture  less  than  i.oo  N.A.  or 
some  of  the  light  from  the  condenser  would  pass  into  the  objective  and  be  trans- 
mitted through  the  microscope  to  the  eye,  and  that  would  destroy  the  dark- field 
effect.  As  oil  immersion  objectives  are  used  in  dark-field  work,  and  practically  all 
of  them  have  an  aperture  ranging  from  the  axis  to  1.20,  1.30  and  1.40  N.A.  at  the 
outer  limit,  some  means  must  be  used  to  bring  the  outer  limit  of  the  aperture  of 
the  objective  below  the  outer  limit  of  the  aperture  of  the  condenser.  To  accom- 


FIG.  48.    PARABOLOID  CONDENSER  FOR  DARK-FIELD  ILLUMINATION. 

Axis    The  principal  optic  axis  of  the  condenser  and  of  the  microscope. 

C-r  Face- view  of  the  top  of  the  condenser  showing  the  centering  ring,  the  spot 
of  white  ink  in  the  middle  and  the  grains  of  starch  scattered  over  the  face  of  the 
condenser  to  aid  in  focusing  high  powers  on  the  centering  ring. 

C-S,  C-S  Sectional  and  face  views  of  the  central  stop  to  cut  out  all  light  below 
an  aperture  of  i.oo  N.A. 

Cover  Glass  The  glass  cover  over  the  object.  For  dry  objectives  it  must  be  of 
the  right  thickness  for  the  objective  (§461).  For  homogeneous  immersion  objec- 
tives the  cover  glass  must  be  less  than  the  working  distance  (§  76). 

Hi,  Hi  Homogeneous  liquid  between  the  condenser  and  the  underface  of  the 
glass  slide,  and  between  the  upper  face  of  the  cover  glass  and  the  objective. 

NA  i.oo  to  NA  1.40.  The  numerical  aperture  of  the  hollow  cone  of  light 
focused  upon  the  object.  This  hollow  cone  ist  represented  by  a  glass  angle  of  67 
to  41  degrees  as  indicated  on  the  left  of  the  condenser  and  on  the  right  above  the 
condenser. 

Objective  The  front  lens  of  the  homogeneous  immersion  objective.  The  light 
rays  deflected  by  the  object  are  indicated  by  white  lines  below  and  through  the 
objective,  and  by  broken  lines  above. 

MM  The  mounting  medium.  It  may  be  of  any  refractive  index  from  air  to 
Canada  balsam. 


CH.  II]    DARK-GROUND    ILLUMINATION  WITH  HIGH  POWERS     73 

plish  this  a  reducing  diaphragm  is  introduced  into  the  objective  cutting  down  the 
aperture  to  less  than  i.oo  N.A.  (fig.  49).  It  would  be  a  convenience  if  homo- 
geneous immersion  objectives  were  made  with  an  aperture  less  than  i.oo  N.A.  to  use 
in  dark-field  work. 

§  125b.  Aperture  of  reducing  diaphragm.  —  While  the  reducing  diaphragm 
for  the  objectives  —  with  an  aperture  above  i.oo  N.A.  —  must  be  of  a  size  to  reduce 
the  aperture  to  less  than  i.oo  N.A.  in  choosing  the  size  it 
must  be  remembered  that  in  dark-field  as  ^in  bright-field 
microscopy  the  resolution  is^  directly  as  the  aperture,  and 
the  brilliancy  with  the  square  of  the  aperture.  Therefore 
the  upper  limit  of  the  aperture  of  the  objective  should  be 
as  great  as  possible  without  endangering  the  loss  of  the 
dark-field  effect.  As  a  result  of  careful  experiments  made 
with  reducing  diaphragms  from  0.40  to  0.97  N.A.  ^dia- 
phragms the  conclusion  was  reached  that  if  but  one 
reducing  diaphragm  is  available,  one  giving  an  aperture  of 
0.80  N.A.  is  the  most  generally  useful,  and  gives  a  dark- 
field  with  practically  all  the  dark-field  condensers  one  is 
likely  to  meet.  If  much  work  on  a  great  variety  of  ob- 
jects and  with  different  sources  of  light  is  to  be  done 
it  would  be  a  great  advantage  to  have  reducing  dia- 
phragms of  0.70,  0.80  and  0.90  N.A.,  then  the  one  which 
gave  the  best  effect  in  a  given  case  could  be  used.  These 
diaphragms  are  easily  introduced  into  the  objective  and  as 
easily  removed.  One  should  bear  in  -mind  that  the  low- 
er end  of  the  diaphragm  must  be  very  close  to  the  back  lens 
of  the  objective.  It  is  also  to  be  remembered  that  when 
the  objective  is  to  be  used  for  the  bright-field  microscopy 
the  diaphragm  should  be  removed  so  that  the  full  benefit 
of  the  aperture  is  secured.  ' 


FIG.  49.  HIGH- 
POWER  OBJECTIVE 
WITH  APERTURE  RE- 
DUCING DIAPHRAGM 
FOR  DARK-GROUND 
ILLUMINATION. 


(From  Chamot.) 

D  Funnel-shaped 
reducing  diaphragm 
screwed  into  the  lower 
end  of  the  "boot" 
opposite  (R). 


§  126.  Immersion  connection  of  condenser 
and  glass  slide.  —  While  the  purpose  of  the 
reflecting  condenser  is  to  produce  a  very 
oblique  beam  of  light  for  illuminating  the  objects,  it  is  evident  at 
once  that  the  laws  of  refraction  will  prevent  the  light  above  a  certain 
angle  from  passing  out  of  the  condenser  to  the  object  unless  the  glass 
slide  is  in  immersion  contact  with  the  condenser.  Figure  50  shows 
the  aperture  of  the  light  which  can  pass  from  the  condenser  when 
air,  water,  glycerin  or  homogeneous  liquid  is  above  it. 


Reasons  for  always  using  homogeneous  immersion.  —  While  it  is  true 
that  the  medium  of  least  refractive  index  determines  the  angle  of  light  that  can 
pass  from  a  denser  to  a  rarer  medium  (fig.  50),  it  is  also  true  that  objects  sur- 
rounded by  air,  water,  etc.,  may  be  in  optical  contact  with  the  glass  slide,  in  which 
case  the  light  will  pass  into  the  object  up  to  the  limit  of  the  refractive  index  of  the 
object,  hence  the  desirability  of  the  light  beam  being  of  great  obliquity,  even  though 
the  object  is  mounted  in  air.  One  can  demonstrate  this  by  cleaning  the  upper 
surface  of  the  condenser  with  the  greatest  kcare,  noting  that  it  is  very  dark 


74     DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    [Cn.  II 

although  the  light  passes  abundantly  into  the  condenser.  It  is  practically  all 
reflected  back  into  the  condenser.  If  now  some  starch  or  flour  is  scattered  over  the 
end  of  the  condenser,  the  particles  in  optical  contact  will  glow  as  if  self-luminous 
(fig. 


§  127.     Slides    and    cover-glasses;     Tube-length    for    dark-field 
microscopy.  —  As  the  object  receives  the  greatest  amount  of  light 


FIG.  50. 


DIAGRAMS  TO  SHOW  THE  REQUISITE  ANGLE  OF  LIGHT  IN  GLASS  TO 
FILL  THE  HEMISPHERE  ABOVE  WITH  LIGHT. 


As  shown  by  the  diagrams  the  numerical  aperture  (NA)  of  the  light  in  the  glass 
in  each  case  must  equal  the  index  of  refraction  (Ir)  of  the  overlying  medium.  Any 
light  above  this  aperture  is  totally  reflected  back  into  the  condenser  (indicated  in 
black). 

a  Glass  with  air  above.  The  light  in  the  glass  must  reach  an  angle  of  41° 
(NA  i.oo)  in  order  to  fill  the  overlying  space  with  light. 

b  Glass  with  water  above.  In  this  case  the  light  in  the  glass  must  reach  an 
angle  of  61°  (NA  1.33)  to  fill  the  hemisphere  above  with  light. 

c  Glass  with  glycerine  (Glyc.)  above.  Here  the  angle  in  the  glass  must  reach 
an  angle  of  75°  15  (NA  1.47)  to  fill  the  overyling  hemisphere  with  light. 

d  Glass  with  homogeneous  liquid  (Horn,  imr.)  above.  As  there  is  no  refrac- 
tion in  this  case  between  the  glass  and  the  overlying  medium,  the  angle  must  be 
90°  (NA  1.52)  in  order  to  fill  the  hemisphere  above  with  light. 


CH.  H]    DARK-GROUND    ILLUMINATION  WITH  HIGH  POWERS    75 


when  it  is  in  the  focus  of  the  condenser,  one  should  know  how  far 
above  the  condenser  the  focus  is  formed,  and  select  slides  of  a  thick- 
ness that  will  allow  the  hollow  light  cone  to  focus  on  the  objects 
mounted  on  the  upper  face  of  the  slide.  In  the  original  dark-field 
condensers,  1856-1877,  and  in  the  more  recent  ones  now  manufactured, 
the  focal  distance  is  carefully  planned,  and  the  manufacturers  state 
on  the  instruments  the 
thickness  of  slide  that  should 
be  used  with  it.  This  thick- 
ness varies  with  different 
manufacturers,  ranging  from 
0.95  mm.  to  1.55  mm.  It  is 
a  good  plan  to  select  several 
of  the  proper  thickness  and 
mark  the  thickness  with  a 
writing  diamond  on  the  end, 
then  one  can  proceed  with 

(Magnified  two  diameters.) 


FIG.  5oa.  UPPER  FACE  OF  THE  FULLY 
LIGHTED  PARABOLOID;  (A)  WITH  CLEAN 
FACE,  (B)  WITH  STARCH  GRAINS  DUSTED 
UPON  IT. 


confidence. 

Cover-glasses  should  be  of 

the  thickness  designated  by  the  makers  for  dry  objectives,  but  for 
oil  immersion  objectives  the  thickness  does  not  matter  provided 
they  are  not  so  thick  that  they  exceed  the  working  distance  of  the 
objective  (§  76-81). 

The  tube-length  of  the  microscope  for  all  objectives  should  be 
adjusted  strictly  in  accordance  with  the  length  given  by  the  manu- 
facturers, as  the  aberrations  are  more  evident  and  troublesome  in 
dark-field  than  in  bright-field  microscopy. 

§  128.  Lighting  for  dark-field  microscopy.  —  As  the  objects 
must  be  seen  by  the  light  they  deflect  into  the  microscope,  and  not 
by  the  light  directly  from  the  source,  it  is  evident  that  the  original 
light  must  be  very  brilliant  for  only  a  small  fraction  of  it  is  finally 
utilized  for  rendering  the  object  visible.  From  the  earliest  use  of 
the  dark-field  method  direct  sunlight  was  found  the  most  satisfactory 
but  as  direct  sunlight  is  available  only  a  part  of  the  time  in  the  most 
favored  regions,  and  is  constantly  shifting  when  available,  artificial 
light  is  mostly  used.  Of  all  the  artificial  lights  available,  the  arc 


76     DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS   [Cn.  II 

light  is  next  in  brilliancy  to  sunlight.  This  also  has  its  drawbacks, 
for  on  account  of  the  burning  away  of  the  carbons  the  light  shifts  and 
requires  frequent  adjustment  of  the  carbons  or  of  the  mirror  or  both. 
The  newly  invented  "Pointolite"  overcomes  the  shifting,  but  is 
available  only  where  direct  current  can  be  had.  The  most  satis- 
factory light  up  to  the  present  is  produced  by  the  "  headlight  " 


FIG.  sob.    SMALL  ARC  LAMP  AND  CONNECTIONS. 
(From  Optic  Projection.) 

A  Small  arc  lamp  base  with  right-angle  carbons  (HC,  VC)  with  insulation 
(In.  In). 

The  arc  and  the  condenser  (C)  are  in  position  to  give  a  parallel  beam. 

Ch  Chimney  over  the  arc,  T,  tube  holding  the  condenser;  sh,  metal  shield 
at  the  end  of  the  condenser  tube. 

Wi      Wire  cable  to  the  lamp  socket  (So)  with  its  key  switch  (K). 

Sp    Separable  plug. 

W  2,  W  3    Wire  to  the  upper  or  horizontal  carbon  (H  C). 

W  4    Wire  to  the  rheostat  (R)  and  to  the  lower  or  vertical  carbon  (VC). 

C    Tips  of  carbons  for  alternating  current. 

D  Tips  of  carbons  for  direct  current,  the  positive  pole  always  being  on  the 
horizontal  carbon  (+),  and  the  negative  pole  on  the  lower  or  vertical  carbon  (— ). 

E  Shield  at  the  end  of  the  condenser  tube;  the  face  of  the  condenser  is  shown 
at  (C). 


CH.  112  DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    ;6a 

lamps  requiring  a  6-volt  current.  This  six-volt  current  may  be 
direct  or  alternating.  In  America  alternating  current  is  more 
commonly  found  in  lighting  systems  than  direct,  but  it  is  at  a  volt- 
age of  no  or  220.  In  order  to  employ  the  alternating  current,  then,  a 


FIG.  5oc.    DIAGRAM  OF  A  LAMP  FOR  DARK-FIELD  MICROSCOPY. 
(About  one  eighth  natural  size.) 

Axis  The  axial  ray  from  the  lamp.  The  parallelizing  lens  is  centered  to  it  and 
the  lamp  filament  is  in  the  focus  of  the  lens. 

bb  Baffle  plates  to  prevent  the  escape  of  light  around  the  bottom  of  the  lamp- 
house. 

DG  Plate  of  daylight  glass  to  which  is  cemented  the  colorless  parallelizing 
lens  (L). 

es    Metal  plate  over  the  lens  to  shade  the  eyes. 

Lamp    The  6-volt,  headlight  lamp  with  very  concentrated  filament. 

Lamp  House.    The  metal  container  for  the  headlight  lamp. 

ms    Mogul  socket  for  the  6-volt  lamp. 

5  Thumb  screw  for  holding  the  lamp  at  the  right  height. 
si    Slide  for  focusing  the  lamp  with  the  parallelizing  lens. 

ot,  it    The  outer  and  inner  tubes  for  centering  the  lamp  vertically. 

Lamp  wires  The  large  conducting  wires  between  the  secondary  of  the  trans- 
former and  the  mogul  socket. 

me  Mistakeless  connection  for  the  lamp  wires  and  the  transformer.  It  is 
unlike  any  connection  for  the  no-volt  circuit  (§  675). 

6  Volts    The  voltage  of  the  secondary  (S)  side  of  the  transformer. 

j/o  Volts.    The  voltage  of  the  supply  to  the  primary  (P)  side  of  the  transformer. 
Transf.    Transformer  to  step  down  from  no  to  6  volts. 
PS    P  the  primary  and  S  the  secondary  side  of  the  transformer. 
Supply  wires.    The  wires  from  the  electric  supply.     The  voltage  in  them  is 
no,  while  that  in  the  lamp  wires  is  6. 


76b  DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    [Ce.  II 

step-down  transformer  must  be  used.  There  is  practically  no  loss 
in  the  transformer  so  that  this  is  an  economical  method.  If  direct 
current  only  is  available  then  it  can  be  used  to  charge  a  storage  bat- 
tery, and  the  current  for  the  6-volt  lamp  drawn  from  the  storage 
battery  yielding  a  six-volt  current.  A  step-down  transformer  cannot 
be  used  with  direct  current,  only  with  alternating. 


FIG.  sod.    HEADLIGHT  LAMP- HOUSE,  WIRING  AND  STEP-DOWN  TRANS- 
FORMER. 

(About  one  eighth  natural  size.) 

D,  Glass  Window  of  daylight  glass  on  the  side  of  the  lamp  house  to  be  used 
for  bright- field  microscopy. 

P.  lens  Parallelizing  lens  of  about  75-mm.  focus.  It  is  cemented  to  a  piece  of 
daylight  glass. 

me  Mistakeless  connection  between  the  lamp  wires  and  those  from  the  sec- 
ondary of  the  transformer.  It  is  different  from  any  connection  for  a  no- volt 
circuit. 

6  Volts.  The  secondary  side  of  the  transformer  where  the  voltage  has  been 
stepped  down  from  no  to  6. 

no  Volts.  The  primary  side  of  the  transformer  where  the  voltage  of  the  cur- 
rent in  the  supply  wires  is  no. 

Transf.    The  transformer,  stepping  the  voltage  down  from  no  to  6. 

The  filament  of  the  6-volt  lamp  is  very  compactly  coiled  so  that  it 
is  about  the  size  of  the  crater  in  an  arc  lamp.  The  lamp  found 
sufficient  for  all  purposes  requires  108  watts.  One  of  72  watts  an- 
swers for  most  purposes,  but  is  not  so  satisfactory  as  the  io8-watt 
one  (fig.  5oc).  As  shown  in  the  illustrations,  the  headlight  lamps 
should  be  enclosed  in  a  lamp-house,  and  have  an  adjustable  fixture 
so  that  they  can  be  centered  with  the  parallelizing  lens  and  also  focused 


CH.  II]  DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS     760 


to  give  a  parallel  beam.     This  requires  vertical  and  horizontal  move- 
ments. 

If  one  does  not  have  available  the  6-volt  lamps  and  a  transformer 
or  the  proper  storage  batteries,  it  is  possible  to  do  much  good  work 
with  a  stereopticon  lamp  of  100  watts 
or  if  the  large  lamp-house  is  availa- 
ble a  2  50- watt  stereopticon  lamp. 
These  can  be  used  on  the  no- volt 
circuit,  alternating  or  direct,  and  can 
be  used  in  either  the  large  lantern 
with  focusing  and  centering  devices 
and  a  parallelizing  lens,  or  the  100- 
watt  stereopticon  lamp  can  be  used 
in  the  Chalet  lamp-house  (fig.  5oe). 

Finally  if  one  has  a  Chalet  or 
other  lamp-house  in  which  the  or- 
dinary, long-necked  C-Mazda  lamp 
is  used  (fig.  37)  one  can  get  fairly 
satisfactory  results;  but  if  much 
work  is  to  be  done  the  io8-watt, 
6-volt  lamp  in  the  centering  and 
focusing  lamp-house  is  much  more 
satisfactory.  As  unmodified  artificial 
light  is  trying  to  the  eyes  of  most 
people,  it  is  desirable  to  employ  the 
daylight  glass  filter  in  dark-field  work. 
As  shown  in  fig.  5oc,  this  is  accomp- 
lished by  cementing,  with  Canada 
balsam,  a  clear,  plano-convex  lens 
of  about  6  centimeters  in  diameter 
and  75-mm.  focus  to  a  piece  of 
polished  daylight  glass.  This  day- 
light effect  enables  most  workers  to  see  minute  details  more  clearly 
than  is  possible  with  unmodified  artificial  light. 

See  also  the  catalogues  of  the  Bausch  &  Lomb  Optical  Co.  and 
of  the  Spencer  Lens  Co.  for  larpps  to  use  in  dark-field  work. 


FIG.  506.  CHALET  LAMP  ix 
SECTION,  WITH  A  IOO-WATT,  MAZDA 
STEREOPTICON  BULB  FOR  DARK- 
FIELD  MICROSCOPY. 

(About  one  sixth  natural  size.) 

c  Electric  cable  to  the  lamp- 
socket  (5). 

dg  Daylight  glass  covering  the 
window  at  the  right. 

ms  loo-watt  Mazda  stereopti- 
con lamp  bulb.  By  means  of  the 
separable  cap  (sc)  and  plug  (sj>) 
this  short-necked  bulb  is  lowered 
so  that  its  tungsten  coil  is  at  the 
same  level  as  the  C-coil  of  the  long- 
necked  bulb  in  fig.  37. 

sc  Socket  cap  partly  separated 
to  show  the  connecting  prongs. 

sp.     Separable  attachment  plug. 

v,v,v    Ventilators. 


;6d    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS   [Ce.  II 

§  129.  Centering  and  focusing  the  dark-field  condenser.  —  While 
it  is  desirable  to  have  the  condenser  well  centered  for  bright-field 
work  it  is  absolutely  essential  in  dark-field  microscopy;  and  all  dark- 
field  condensers  worthy  of  the  name  have  centering  arrangements. 
To  center  the  condenser  after  it  is  put  in  the  substage  ring  and  pushed 
up  so  that  the  upper  end  of  the  condenser  is  flush  with  the  upper  sur- 
face of  the  stage,  or  very  slightly  above  it,  one  uses  a  low  objective 
and  ocular  and  focuses  down  until  the  top  of  the  condenser  is  in 
focus.  There  is  a  delicate  ring  on  the  upper  face  of  the  dark-field 
condenser;  this  must  be  made  to  occupy  the  exact  center  of  the  field 
by  using  the  centering  screws  of  the  condenser  mounting.  This 
centering  answers  for  most  work,  but  to  center  the  homogeneous 
immersion  objective,  or  other  high- power  objective,  and  the  condenser 
where  the  field  is  so  small  that  the  ring  on  the  condenser  is  consider- 
ably larger  than  the  field  of  the  objective,  one  can  make  a  minute 
dot  of  white  ink  in  the  exact  center  of  the  upper  face  of  the  condenser 
(fig.  48c,  r).  A  crow-quill  pen  is  good  for  this.  Then  some  starch 
or  flour  is  scattered  over  the  face  of  the  condenser  so  that  one  can 
focus  upon  it  with  the  high  power.  For  the  centering  the  oil-immer- 
sion objective  it  is  not  necessary  to  use  any  immersion  liquid.  The 
condenser  is  then  got  as  nearly  centered  as  possible  with  the  low 
power,  and  then  the  high  power  is  focused  upon  the  condenser  and  it 
is  shifted  if  necessary  until  the  central  spot  of  ink  is  in  the  middle  of 
the  field  of  the  objective.  The  spot  of  white  ink  can  be  easily  re- 
moved by  the  use  of  a  moist  cloth  or  a  moist  piece  of  lens  paper. 

Focusing  the  condenser  on  the  specimen  is  accomplished  thus: 
A  slide  of  the  thickness  required  by  the  condenser  is  used.  On  it 
is  mounted  some  starch  in  water,  and  a  cover-glass  is  added.  A 
drop  of  cedar  oil  is  put  on  the  top  of  the  condenser  and  on  the  under- 
side of  the  slide  opposite  the  preparation.  The  slide  is  then  put 
in  place  and  the  condenser  moved  up  if  necessary  until  the  top  almost 
touches  the  slide.  If  now  one  lights  well  and  examines  the  specimen 
with  a  low  power  there  will  be  seen  a  spot  or  ring  of  light.  By  mov- 
ing the  condenser  up  and  down  one  can  find  the  position  where  the 
spot  of  light  on  the  specimen  is  the  smallest  and  brightest,  and  that 
will  be  the  position  of  the  best  focus,  and  where  the  most  favorable 


CH.  II]    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS     766 

light  for  observation  will  be  obtained.  If  the  condenser  is  consid-' 
erably  too  high  or  too  low  the  object  will  be  lighted  with  a  ring  of 
light  with  a  dark  center.  It  is  only  when  the  hollow  cone  of  light  is 
focused  on  the  specimen  that  the  entire  field  will  be  equally  bright. 
After  a  little  practice  one  can  focus  the  condenser  on  the  specimen  to 
be  studied .  The  image  is  so  much  more  satisfactory  that  it  is  worth 
the  trouble. 

§  130.  Getting  the  light  to  pass  up  through  the  condenser. — The 
microscope  mirror  should  be  about  20-30  cm.  from  the  arc  lamp  or 
from  the  io8-watt  daylight  lantern  (fig.  5oc,  d).  It  must  be  quite 
close  to  the  stereopticon  lamp  (fig.  500) .  One  can  tell  the  best  posi- 
tion by  holding  a  piece  of  white  paper  in  the  parallel  beam  to  find  the 
position  where  the  light  is  most  uniform,  and  brightest.  Place  the 
microscope  so  that  the  parallel  beam  strikes  the  mirror  at  this  bright- 
est point.  The  mirror  will  be  nearly  filled  with  the  light.  If  it  is 
difficult  to  see  on  the  mirror  one  can  use  some  white  paper  over  the 
mirror.  Sometimes  the  lamp-house  must  be  tipped  forward,  or 
the  microscope  raised  or  tipped  backward,  to  bring  the  light  beam 
and  mirror  in  proper  relation.  Now  turn  the  mirror  so  that  the  light 
will  be  turned  up  into  the  condenser.  If  the  condenser  has  a  drop 
of  cedar  oil  on  it  or  if  a  slide  is  in  immersion  contact  and  a  drop  of 
cedar  oil  is  on  the  cover-glass  one  can  see  when  the  light  passes  through 
the  condenser.  If  now  the  oil  immersion  is  focused  on  the  object  it 
usually  takes  but  a  slight  manipulation  of  the  mirror  to  get  the  best 
light.  One  should  never  be  satisfied  until  the  light  is  at  its  best,  and 
this  can  only  be  told  by  considerable  experimenting.  There  is  an- 
other sign  which  gives  great  help,  and  is  particularly  applicable  in 
the  evening  or  in  a  dimly  lighted  room.  The  light  which  passes  from 
the  lamp  to  the  mirror  and  then  to  the  condenser  passes  on  to  the  top 
of  the  condenser  where  the  lamp-filament  is  nearly  at  a  focus.  From 
the  top  of  the  condenser  there  is  always  a  considerable  reflection  and 
this  reflection  passes  back  through  the  condenser  to  the  mirror  and 
then  back  toward  the  lamp-house.  Wherever  it  strikes  will  appear 
a  spot  of  light  (fig.  sob,  e).  If  now  the  mirror  is  turned  slightly  until 
this  spot  of  light  is  thrown  into  the  parallelizing  lens  the  light  will 
also  be  very  satisfactory  on  the  specimen.  After  a  little  practice 
one  can  light  the  microscope  with  great  certainty  by  the  aid  of  this 


76f    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    [Cn.  II 

spot  of  light  giving  the  indication  when  the  mirror  is  in  the  right 
position.  Slight  changes  only  will  then  be  needed  on  looking  into 
the  microscope  to  get  the  best  effect. 

§  131.  Practical  application  of  dark-field  microscopy.  —  In  the 
practical  application  of  dark-field  microscopy  it  is  almost  self-evident 
that  it  can  be  used  successfully  only  with  scattered  objects,  that  is 
objects  with  blank  space  between  them.  If  the  objects  covered  the 
whole  field,  then  the  field  would  all  be  bright,  and  there  would  be 
no  dark-ground  effect.  It  is  not  then  applicable  to  the  study  of 
microscopic  sections,  for  they  cover  many  times  over  the  entire  field 
with  high  powers.  But  in  Biology,  using  the  word  in  the  compre- 
hensive sense  employed  by  Huxley,  it  is  applicable  and  will  also 
surely  give  much  information  in  the  following  cases: 

04)  In  the  study  of  unicellular  organisms  in  both  the  plant  and 
the  animal  kingdoms  (i.e.,  bacteria  and  all  other  plant  micro-organ- 
isms, and  the  animal  micro-organism). 

(B)  In  the  study  of  multicellular  organisms  among  both  plants  and 
animals  it  is  especially  applicable  to  their  fluid  parts,  and  to  their 
individual  cells  when  properly  isolated.  In  the  vertebrates,  in- 
cluding man,  tnis  would  apply  especially  to  the  blood  and  lymph 
with  their  granular  contents,  the  tissue  fluids  and  the  fluids  of  the 
natural  spaces  like  the  pleural  and  pericardial  cavities,  the  peri- 
toneal cavity,  and  the  liquids  found  in  the  cavities  of  the  central  ner- 
vous system,  the  joint  cavities  and  the  tendon  sheaths.  It  is  also 
of  great  help  in  the  study  of  the  liquids  found  in  mucous  containers, 
as  milk,  bile,  urine,  saliva  and  indeed  in  the  study  of  all  the  liquids 
of  the  body. 

Dr.  Chamot  points  out  its  help  in  the  study  of  foods,  fibers,  crys- 
tallization phenomena,  sub-microscopic  particles  and  colloids.  He 
adds  further  (p.  40):  "  This  method  is  invaluable  for  demonstrating 
the  presence  of  very  minute  bodies  or  those  whose  index  of  refraction 
is  so  nearly  the  same  as  that  of  the  medium  in  which  they  occur  as 
to  cause  them  to  escape  detection  when  illuminated  by  transmitted 
light,"  i.e.,  the  ordinary  light  used  for  bright-field  microscopy. 

§  132.  Summary  of  steps  necessary  for  successful  dark-field 
observation.  —  (i)  A  powerful  source  of  light  must  be  provided. 


CH.  II]    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    y6g 

(2)  The  dark-field  condenser  must  be  put  into  the  ring  of  the  sub- 
stage,  and  raised  until  its  top  is  flush  with  the  upper  surface  of  the 
stage.     The  condenser  is  then  accurately  centered.     If  there  is  an 
iris  diaphragm  below  the  condenser  it  should  be  made  wide  open. 

(3)  A  homogeneous  immersion  objective,    containing  a    suitable 
reducing  diaphragm  (i.e.,  one  giving  0.80  to  0.90  N.A.),  is  screwed 
into  the  opening  in  the  nose-piece  of  the  microscope. 

(4)  Slides  and  cover-glasses  of  the  proper  thickness  are  cleaned 
thoroughly  and  put  in  position  for  rapid  handling. 

(5)  The  preparation  to  be  examined  —  blood,  saliva,  etc.  —  is  put 
on  the  slide  and  covered.     The  cover-glass  is  sealed  with  mineral  or 
with  castor  oil,  or  with  shellac  cement  and  the  slide  is  labeled. 

(6)  A  drop  of  homogeneous  oil  is  put  upon  the  upper  face  of  the 
condenser  and  one  or  more  drops  on  the  under  side  of  the  slide  op- 
posite the  preparation.     One  or  more  drops  are  then  put  on  the 
cover-glass.     Plenty  of  oil  is  used  so  that  when  the  slide  is  moved 
around  in  the  examination  good  immersion  contact  will  be  maintained. 

(7)  The  slide  is  placed  on  the  stage  so  that  the  oil  makes  immer- 
sion contact  with  the  underside  of  the  slide  and  the  top  of  the  con- 
denser.    The  condenser  may  need  to  be  raised  slightly  to  make  the 
contact  perfect,  or  it  may  need  to  be  lowered  slightly  so  that  the  slide 
will  rest  on  the  stage. 

(8)  The  microscope  and  lamp  are  arranged  to  give  the  best  light 
by  having  the  parallel  beam  fall  on  the  mirror,  then  the  mirror  is 
turned  slightly  until  there  appears  a  brilliant  point  of  light  in  the  oil 
on  the  cover-glass.     The  objective  is  then  lowered  until  it  dips  into 
the  oil. 

(7)  The  microscope  is  then  focused  and  the  light  made  as  brilliant 
as  desired  by  slightly  moving  the  mirror.  In  focusing  it  will  be  seen 
that  when  the  objective  is  near  the  object  the  whole  field  will  look 
bright,  but  as  the  focus  is  reached  the  specimen  alone  will  be  bright 
and  the  field  dark. 

(10)  Dark-field  microscopy  requires  more  skill  and  accuracy  of 
manipulation  than  does  bright-field  microscopy,  but  the  increased 
visibility  pays  for  all  the  trouble  required.  It  is  best  to  work  in  the 
evening  or  in  a  dark  or  dimly  lighted  room  for  then  the  eyes  are  ad- 


76h    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    [Cn.  U 

justed  for  twilight  vision  and  can  most  easily  make  out  the  finest 
details. 

§  132a.  Example  illustrating  the  method  of  procedure  in  dark-field  micro- 
scopy. —  Perfectly  fresh  blood  is  one  of  the  best  objects  to  study  by  this  method. 
As  pointed  out  by  Dr.  Edmunds  nearly  50  years  ago,  blood  with  the  dark- field 
illumination  appears  like  a  new  object  so  many  things  are  seen  with  the  greatest 
distinctness  that  are  wholly  invisible  or  merely  glimpsed  when  examined  by  the 
bright- field  method. 

(1)  Carefully  cleaned  slides  and  cover-glasses  of  the  right  thickness  are  placed 
where  they  can  be  easily  grasped. 

(2)  For  obtaining  the  fresh  blood  the  part  to  be  punctured  should  be  cleaned 
well  with  95  %  alcohol  and  then  with  a  sterilized  needle  or  Dr.  Moore's  Haemos- 
past,  the  puncture  is  made.     The  drop  of  blood  exuding  can  be  quickly  touched 
by  a  cover-glass,  and  the  cover  put  on  the  center  of  one  of  the  prepared  slides. 
If  a.  small  amount  adheres  to  the  cover,  it  will  spread  out  in  a  very  thin  layer  when 


corpuscles 

after  the  preparation  is  on  the  slide. 

If  all  the  preparations  are  quite  red,  after  a  few  minutes,  one  can  be  made 
thinner  by  pressing  firmly  on  the  cover  by  the  ball  of  the  thumb  covered  with  gauze 
or  lens  paper.  The  gauze  or  paper  absorbs  the  blood  which  runs  out  at  the  edge 
of  the  cover.  In  order  to  prevent  evaporation  and  to  help  anchor  the  cover- 

tlass  so  that  it  will  not  move  by  the  pull  of  the  viscid  homogeneous  immersion 
uid,  it  is  advisable  to  seal  the  cover  by  painting  a  ring  of  liquid  vaseline  (petro- 
leum oil)  or  castor  oil  around  the  edge  of  the  cover.  One  of  the  thick  preparations 
should  not  be  sealed,  but  kept  for  irrigation  with  normal  salt  to  show  especially 
the  fibrin  network.  When  ready  to  study  the  blood,  put  a  large  drop,  or  two  large 
drops,  of  homogeneous  liquid  on  the  underside  of  the  slide  directly  opposite  the 
specimen,  and  place  the  slide  on  the  stage  of  the  microscope  so  that  the  immer- 
sion liquid  will  come  over  the  face  of  the  condenser.  Then  a  drop  of  immersion 
liquid  is  put  on  the  cover-glass  and  the  objective  run  down  into  it.  If  the  light- 
ing is  secured  as  explained  above  one  soon  learns  to  focus  on  the  specimen.  In 
general,  the  field  all  looks  bright  just  before  the  objective  gets  down  to  the  level 
for  seeing  the  specimen. 

(a)  The  erythrocytes  will  appear  like  dark  discs  with  bright  rims  owing  to  the 
convex  borders. 

(b)  The  leucocytes  appear  as  real  white  corpuscles  owing  to  the  granules  within 
them  which  turn  the  light  into  the  microscope.     If  the  room  is  moderately  warm 
—  20°  C.  or  more  —  the  leucocytes,  some  of  them,  will  undergo  the  amoeboid 
movement,  and  the  picture  they  present  will  be  a  revelation  to  those  who  never 
saw  it  or  only  with  the  bright-field  microscope.     From  the  clearness  with  which 
everything  can  be  seen  the  minutest  change  can  be  followed,  and  also  the  most 
delicate  pseudopod  detected.     Another  striking  feature  will  be  noticed  in  the 
moving  ones,  that  is,  the  vigorous  Brownian  movement  of  the  granules  in  the 
part  of  the  leucocyte  with  the  amoeboid  movement.     In  those  showing  no  amoe- 
boid movement  there  is  usually  no  sign  of  the  Brownian  movement  of  the  granules; 
also  if  a  part  of  the  leucocyte  is  not  undergoing  amoeboid  movement  the  particles 
in  it  are  usually  motionless. 

(c)  The  fibrin  network  will  be  seen  like  a  delicate  cob-web  between  the  cor- 
puscles.    In  different  parts  of  the  specimen  one  can  find  all  the  appearances  of  the 
fibrin  shown  in  text  books  on  the  blood. 


CH.  II]    DARE-GROUND  ILLUMINATION  WITH  HIGH  POWERS     y6i 


(d)  Chylomicrons  appear  everywhere  like  bright  points  in  the  empty  spaces 
between  the  corpuscles.  They  are  in  every  active  Brownian  or  pedetic  move- 
ment. These  chylomicrons  will  probably  be  the  most  unusual  part  to  those  study- 
ing blood  with  the  dark  field  for  the  first  time.  The  term  Chylomicron  is  from 
two  Greek  words,  xO\6$  (chylos)  juice  or  chyle,  and  fj.tnp6v  (micron),  any  small 


TASTING 


CMYLOMICPONS 


FIBRIN 


UCOCYTCS 
THDOMBOCYTCS 
COYTMBOCYTES 


TASTING. 


E  BYTttBOCYTtS 


FIG.  sof.    FRESH  BLOOD  WITH  BRIGHT-AND  WITH  DARK-FIELD. 
(x  400  DIAMETERS.) 

In  the  illustration  the  chylomicrons,  the  fibrin,  the  thrombocytes  and  leucocytes 
are  too  definite  in  the  bright  field,  but  their  appearance  is  not  exaggerated  in  the 
dark  field. 

thing.  In  modern  metrology  it  signifies  the  millionth  of  a  meter  (§  246).  I  have 
introduced  this  word  to  show  the  origin  of  these  bodies  from  the  chyle,  and  to  in- 
dicate their  average  size.  In  1840-1842,  Gulliver  called  these  minute  granules  the 
"molecular  base  of  the  chyle"  and  showed  that  they  were  identical  in  the  thoracic- 
duct  and  in  the  blood  vessels  of  the  same  animal.  He  gave  their  average  size  as 


76j    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    [Cn.  II 

i  /  36,000  to  i  /  24,oooth  of  an  inch.  They  have  been  called  by  others  free  granules 
or  granulations,  elementary  particles,  etc. 

(e)  A  very  striking  view  of  the  fibrin  network  may  be  obtained  by  irrigating 
the  thick  blood  preparation.  If  a  drop  of  normal  salt  solution  is  placed  on  one 
edge  of  the  cover-glass  and  a  piece  of  blotting  paper  on  the  other  the  liquid  is 
drawn  through,  washing  out  many  of  the  erythrocytes.  If  the  washing  out  proc- 
ess is  watched  under  the  microscope  the  erythrocytes  will  be  seen  gliding  over 
or  through  the  fibrin  network,  or  some  of  them  will  be  anchored  at  one  end  and 
if  the  current  is  rapid  the  corpuscles  will  be  pulled  out  into  pear-shaped  forms. 

The  leucocytes  look  like  big  white  boulders  in  the  stream,  wholly  unmoved  by 
the  rushing  torrent  around  them. 

§  132b.  Ultramicroscopy.  —  In  ultra-  as  in  dark-field  microscopy  the  objects 
seem  to  be  self-luminous  in  a  dark  space  or  field. 

Dark- field  microscopy  deals  with  relatively  large  objects,  0.2  fj.  or  more  in  dia- 
meter, that  is,  those  which  come  within  the  resolving  power  of  the  microscope. 
Ultramicroscopy  on  the  other  hand  deals  with  objects  so  small  that  they  do  not 
show  as  objects  with  details  but  one  infers  their  presence  by  the  points  of  light 
which  they  deflect  into  the  microscope.  This  can  be  made  clear  by  an  easily 
tried  naked-eye  experiment.  Suppose  one  is  in  a  dark  room  and  a  beam  of  sun- 
light or  electric  light  is  admitted.  Unless  one  is  in  the  path  of  this  beam  it  will 
remain  invisible,  but  if  there  are  particles  of  vapor  or  dust  in  the  room  they  will 
deflect  the  light  and  no  matter  where  one  stands  they  will  appear  as  shining  par- 
ticles. The  character  of  the  particles  cannot  be  made  out,  but  the  light  which 
they  deflect  enables  the  observer  to  infer  their  presence. 

Dark-field  and  Ultramicroscopy  are  said  to  merge  into  each  other  because,  in 
studying  specimens  like  saliva,  etc.,  some  of  the  elements  are  relatively  large  and 
show  details,  while  others  are  so  small  that  they  show  simply  as  points  of  light 
with  diffraction  discs.  The  larger  objects  showing  details  or  microscopic  resolu- 
tion, come  within  the  province  of  the  dark-field  microscope,  while  the  smallest  ones 
are  in  the  province  of  the  ultramicroscope.  With  such  specimens  one  might  with 
equal  propriety  call  the  instrument  used  for  examining  them  an  ultra  microscope 
or  a  dark-field  microscope  depending  on  whether  the  attention  was  directed  toward 
the  smallest  objects  or  toward  the  larger  ones. 

COLLATERAL  READING  FOR  CHAPTER  II 

BEALE,  L.  S.  —  How  to  work  with  the  microscope,  pp.  26-29. 

BECK,  CONRAD.  —  Cantor  lectures,  Roy.  Soc.  Arts,  1907. 

CARPENTER,   WM.   B.  —  The  microscope  and  its  revelations,  first  edition,  1856. 

Excellent  discussion  of  dark-field  microscopy;   also  in  the  6th  edition,  1881. 
CARPENTER-DALLINGER.  —  The    microscope    and    its    revelations,    8th    edition, 

1901. 
CHAMOT,   E.    M.  —  Elementary   chemical   microscopy.     Excellent   discussion   of 

the  dark-field  and  ultra-microscope. 
EDMUNDS,  JAMES.  —  On  a  new  paraboloid  illuminator.     Monthly  Microscopical 

Journal,  Vol.  xviii,  1877,  pp.  78-85. 
GAGE,  S.  H.  —  Dark- field  microscopy  and  the  history  of  its  development.     Trans. 

Amer.  Micr.  Soc.,  Vol.  XXXIX,  1920,  pp.  95-141- 
NELSON,  E.  M.  —  The  substage  condenser.     Jour.  Roy.  Micr.  Soc.;     Vol.  XI, 

1891,  pp.  90-105. 


CH.  II]    DARK-GROUND  ILLUMINATION  WITH  HIGH  POWERS    ;6k 

"Pointolite"  lamp  for  direct  current.  Developed  by  E.  A.  Gimingham  and 
S.  R.  Milliard  at  the  Edison  Swan  Elect.  Co.,  Ltd.  Ponders  End,  Middle- 
sex, England.  This  is  a  tungsten  enclosed  arc  for  direct  current.  It  yields 
about  500  candlepower,  and  has  a  life  of  500  hours.  See  Trans.  Inst.  Elect. 
Engineers,  Vol.  LIV,  1916,  p.  15;  Illuminating  Engineer,  Vol.  II,  p.  324, 
1919;  Chem.  and  Metallurgical  Enginering,  Vol.  XXII,  1920,  p.  281. 
SPITTA,  E.  J.  —  Microscopy,  the  construction,  theory  and  use  of  the  microscope 

(1907). 

WENHAM,  F.  H.  —  (Dark-field  Illumination.)    Trans.  Micr.  Soc.  London,  Vol.  Ill, 
1850,  pp.  83-90;   Quart.  Jour.  Micr.  Sci.,  Vol.  II,  1854,  pp.  145-158;  Trans. 
Micr.  Soc.,  London,  in  Quart.  Jour.  Micr.  Sci.,  Vol.  IV,  1856,  pp.  55-60. 
WRIGHT,  SIR  A.  E.  —  The  Principles  of  microscopy  (1907). 


CHAPTER  III 

ADJUSTABLE  AND  IMMERSION  OBJECTIVES;  REFRACTION  AND 
COLOR  IMAGES;  BINOCULAR  MICROSCOPES;  CARE  OF  THE 
MICROSCOPE;  CARE  OF  THE  EYES;  WORK  TABLES;  TEST- 
ING THE  MICROSCOPE;  MARKERS  AND  MECHANICAL 
STAGES;  ROYAL  MICROSCOPICAL  SOCIETY  STANDARDS 

§  133.    Apparatus  and  material  for  Chapter  III. 

1.  Compound  microscope  with  dry,  7.  Histological  specimens  like  mus- 
Wfcter,  and  homogeneous   immersion       cle  fibers,  etc. 

objectives.  8.  Cedar   oil,    xylene,    chloroform, 

2.  Simple    microscope.  glycerin,  and  lens  paper. 

3.  Eye  shade  (fig.  56).  9.  Binocular    microscope. 

4.  Shield  for  microscope  and  ob-  10.  Opaque    objects    like    insects, 
server  (fig.  33).  feathers,  etc. 

5.  Slides    and    cover-glasses    (Ch.  n.  Mounted  fly's  wing. 

X).  12.  Markers,     mechanical     stages, 

6.  Pleurosigma  (§  115)  and  stained       colored     shellac     and     camel's     hair 
bacteria.  brush  for  pointers. 

ADJUSTABLE,   WATER  AND  HOMOGENEOUS  IMMERSION  OBJECTIVES 

Experiments 

§134.  Adjustment  for  objectives.  —  As  stated  above  (§31)  the 
aberration  produced  by  the  cover-glass  (fig.  51)  is  compensated  for 
by  giving  the  combinations  in  the  objective  a  different  relative  posi- 
tion than  they  would  have  if  the  objective  were  to  be  used  on  uncov- 
ered objects.  Although  this  relative  position  cannot  be  changed  in 
unadjustable  objectives,  one  can  secure  the  best  results  of  which  the 
objective  is  capable  by  selecting  covers  of  the  thickness  for  which  the 
objective  was  corrected.  (See  table,  Ch.  IX.)  Adjustment  may  be 
made  also  by  increasing  the  tube -length  for  covers  thinner  than  the 
standard  and  by  shortening  the  tube-length  for  covers  thicker  than  the 
standard. 

In  learning  to  adjust  objectives,  it  is  best  for  the  student  to  choose 

77 


ADJUSTMENT  OF  OBJECTIVES 


[CH.  Ill 


:  Slide— 


FlG.   51. 


ABERRATION  PRODUCED  BY  THE 
COVER-GLASS. 


The  extension  of  the  principal  optic 

The  cover-glass. 

from    the 


some  object,  like  Pleurosigma  (§  115),  whose  structure  is  well  agreed 
upon,  and  then  to  practise  lighting  it,  shading  the  stage  and  adjusting 
the  objective,  until  the  proper  appearance  is  obtained.  The  adjust- 
ment is  made  by  turning  a  ring  or  collar  which  acts  on  a  screw  and 

increases  or  diminishes  the 

|   i  /  2/  a/  distance  between  the  sys- 

tems of  lenses,  usually 
the  front  and  the  back 
systems  (fig.  35). 

§  135.  Directions  for 
adjustment.  —  (i)  The 
thicker  the  cover-glass  the 
closer  together  are  the  sys- 
tems brought  by  turning 
the  adjusting  collar  from 
the  zero  mark  and  con- 
versely; (2)  the  thinner 
the  cover-glass,  the  further 
must  the  systems  be  sep- 
arated, i.e.,  the  adjusting 
collar  is  turned  nearer  the 
zero  or  the  mark  "  un- 
covered." This  also  in- 
creases the  magnification  of  the  objective  (§  235). 

The  following  specific  directions  for  making  the  cover-glass  adjust- 
ment are  given  by  Mr.  Wenham  (Carpenter,  yth  Ed.,  p.  166) :  "  Select 
any  dark  speck  or  opaque  portion  of  the  object,  and  bring  the  outline 
into  perfect  focus;  then  lay  the  finger  on  the  milled-head  of  the  fine 
motion  and  move  it  briskly  backwards  and  forwards  in  both  direc- 
tions from  the  first  position.  Observe  the  expansion  of  the  dark  out- 
line of  the  object,  both  when  within  and  when  without  the  focus.  If 
the  greater  expansion  or  coma  is  when  the  object  is  without  the  focus, 
or  farthest  from  the  objective  [i.e.,  in  focusing  up],  the  lenses  must 
be  placed  further  asunder,  or  toward  the  mark  uncovered  [the  adjust- 
ing collar  is  turned  toward  the  zero  mark,  as  the  cover-glass  is  too  thin 
for  the  present  adjustment].  If  the  greater  expansion  is  when  the 


Axis 
axis. 

Cover 

1,2,3     Three   rays   originating 
object  mounted  in  balsam. 

r,  r,  r  Points  of  refraction  as  the  three 
rays  emerge  from  the  upper  surface  of  the 
cover  into  the  air. 

O     Object  from  which  the  rays  originate. 

i,  2, 3  The  three  levels  from  which  the  rays 
seem  to  originate  when  traced  backward  from 
their  points  of  emergence.  This  gives  the 
effect  of  spherical  aberration  (Ch.  IX). 


CH.  Ill]  ADJUSTMENT  OF  OBJECTIVES  79 

object  is  within  the  focus,  or  nearest  the  objective  [i.e.,  in  focusing 
down],  the  lenses  must  be  brought  closer  together,  or  toward  the  mark 
covered  [i.e.,  the  adjusting  collar  should  be  turned  away  from  the 
zero  mark,  the  cover-glass  being  too  thick  for  the  present  adjustment]." 
In  most  objectives  the  collar  is  graduated  arbitrarily,  the  zero  (o) 
mark  representing  the  position  for  uncovered  objects.  Other  objec- 
tives have  the  collar  graduated  to  correspond  to  the  various  thickness 
of  cover-glasses  for  which  the  objective  may  be  adjusted.  This  seems 
to  be  an  admirable  plan ;  then  if  one  knows  the  thickness  of  the  cover- 
glass  on  the  preparation  (Chs.  IX-X)  the  adjusting  collar  may  be  set 
at  a  corresponding  mark,  and  one  will  feel  confident  that  the  adjust- 
ment will  be  approximately  correct.  It  is  then  only  necessary  for 
the  observer  to  make  the  slight  adjustment  to  compensate  for  the 
mounting  medium  or  any  variation  from  the  standard  length  of  the 
tube  of  the  microscope.  In  adjusting  for  variations  of  the  length  of 
the  tube  from  the  standard  it  should  be  remembered  that:  (i)  If 
the  tube  of  the  microscope  is  longer  than  the  standard  for  which  the 
objective  was  corrected,  the  effect  is  approximately  the  same  as  thick- 
ening the  cover-glass,  and  therefore  the  systems  of  the  objective  must 
be  brought  closer  together,  i.e.,  the  adjusting  collar  must  be  turned 
away  from  the  zero  mark.  (2)  If  the  tube  is  shorter  than  the  standard 
for  which  the  objective  is  corrected,  the  effect  is  approximately  the 
same  as  diminishing  the  thickness  of  the  cover-glass,  and  the  systems 
must  therefore  be  separated  (fig.  35),  i.e.,  turned  toward  the  zero 
mark. 

In  using  the  tube- length  for  cover  correction  shorten  the  tube  for 
too  thick  covers,  and  lengthen  the  tube  for  too  thin  covers. 

Furthermore,  whatever  the  interpretation  by  different  opticians 
of  what  should  be  included  in  tube-length,  and  the  exact  length  in 
millimeters,  its  importance  is  very  great,  for  each  objective  gives  the 
most  perfect  image  of  which  it  is  capable  with  the  tube-length  for 
which  it  is  corrected,  and  the  more  perfect  the  objective  the  greater 
the  ill-effects  on  the  image  of  varying  the  tube-length  from  the  stand- 
ard. The  plan  of  designating  exactly  what  is  meant  by  tube-length 
and  engraving  on  each  objective  the  tube-length  for  which  it  is  cor- 
rected, is  to  be  commended,  for  it  is  manifestly  difficult  for  each  worker 


8o  REFRACTION  AND   COLOR  IMAGES  [CH.  Ill 

with  the  microscope  to  find  out  for  himself  for  what  tube-length  each 
of  his  objectives  was  corrected  (see  Ch.  IX). 

§  136.  Water  immersion  objectives.  —  Put  a  water  immersion 
objective  in  position  (§  44)  and  the  fly's  wing  for  object  under  the 
microscope.  Place  a  drop  of  distilled  water  on  the  cover-glass,  and 
with  the  coarse  adjustment  lower  the  tube  till  the  objective  dips  into 
the  water,  then  light  the  field  well  and  turn  the  fine  adjustment  one 
way  and  another  till  the  image  is  clear.  Water  immersions  are  ex- 
ceedingly convenient  in  studying  the  circulation  of  the  blood,  and  for 
many  other  purposes  where  aqueous  liquids  are  liable  to  get  on  the 
cover-glass.  If  the  objective  is  adjustable,  follow  the  directions  given 
in  §  135. 

When  one  is  through  using  a  water  immersion  objective,  remove 
it  from  the  microscope  and  with  some  lens  paper  wipe  all  the  water 
from  the  front  lens.  Unless  this  is  done  dust  collects  and  sooner  or 
later  the  front  lens  will  be  clouded.  It  is  better  to  use  distilled  water 
to  avoid  the  gritty  substances  that  are  liable  to  be  present  in  natural 
water,  as  these  gritty  particles  might  scratch  the  front  lens. 

REFRACTION  AND  COLOR  IMAGES 

• 

§  137.  Refraction  images  are  those  mostly  seen  in  studying  micro- 
scopic objects.  —  They  are  the  appearances  produced  by  the  refrac- 
tion of  the  light  on  entering  and  on  leaving  an  object.  They  therefore 
depend  (a)  upon  the  form  of  the  object,  (b)  upon  the  relative  refrac- 
tive powers  of  object  and  mounting  medium.  With  such  images  the 
diaphragm  should  not  be  too  large  (see  §  106-107). 

If  the  color  and  refractive  index  of  the  object  were  exactly  like  the 
mounting  medium,  it  could  not  be  seen.  In  most  cases  both  refractive 
index  and  color  differ  somewhat;  there  is  then  a  combination  of  color 
and  refraction  images  which  is  a  great  advantage.  This  combina- 
tion is  generally  taken  advantage  of  in  histology.  The  air  bubble  in 
§  194  is  an  example  of  a  purely  refractive  image. 

A  purely  refractive  image  like  that  given  by  an  air  bubble  or  a  fat 
globule  gives  a  dark  border  for  central  transmitted  light,  and  a  light 
border  on  a  black  field  with  very  oblique  light,  such  as  is  given  by  the 
mirror  turned  far  to  one  side  or  by  a  central  stop  when  the  condenser 


CH.  Ill]  REFRACTION  AND   COLOR  IMAGES  81 

is  used  (§  123,  201).  In  both  cases  the  object  is  in  outline.  As 
pointed  out  by  Wright  (p.  5,  41)  the  visibility  of  the  object  shown  in 
outline  depends  on  the  width  of  the  outline  and  not  on  the  diameter 
of  the  whole  object.  If  the  width  of  the  outline  is  too  narrow  to  in- 
clude the  necessary  visual  angle  of  i  minute  (§  227)  the  whole  object 
fades  into  the  background  and  is  no  longer  visible.  On  the  other 
hand,  if  the  object  is  colored,  then  it  is  visible  so  long  as  its  entire 
diameter  gives  a  visual  angle  of  i  minute  or  more. 

One  can  see  from  the  above  what  a  tremendous  advantage  it 
is  in  studying  the  finest  details  of  structure  to  have  them  brilliantly 
colored. 

HOMOGENEOUS  IMMERSION  OBJECTIVES 
Experiments 

As  stated  above  (§  25),  these  are  objectives  (fig.  2iB)  in  which  a 
liquid  of  the  same  refractive  index  as  the  front  lens  of  the  objective 
is  placed  between  the  front  lens  and  the  cover-glass. 

§  138.  Refraction  images.  —  Put  a  2  mm.  homogeneous  immersion 
objective  in  position;  employ  a  condenser.  Use  some  histological 
specimen  like  a  muscular  fiber  as  object;  make  the  diaphragm  open- 
ing about  9  mm.  in  diameter,  add  a  drop  of  the  homogeneous  immer- 
sion liquid,  and  focus  as  directed  in  §  72.  The  object  will  be  clearly 
seen  in  all  its  details  by  the  unequal  refraction  of  the  light  traversing 
it.  The  difference  in  color  between  it  and  the  surrounding  medium 
will  also  increase  the  sharpness  of  the  outline.  If  an  air  bubble  prepa- 
ration (§  195)  were  used,  one  would  get  pure  refraction  images. 

§  139.  Color  images.  —  Use  some  stained  bacteria  as  Bacillus 
tuberculosis  for  object.  Put  a  drop  of  the  immersion  liquid  on  the 
cover-glass  or  on  the  front  lens  of  the  homogeneous  objective.  Re- 
move the  diaphragms  from  the  illuminator  or  in  case  the  iris  diaphragm 
is  used,  open  it  to  its  greatest  extent.  Focus  the  objective  down  so 
that  the  immersion  fluid  is  in  contact  with  both  the  front  lens  and  the 
cover-glass;  then  with  the  fine  adjustment  get  the  bacteria  in  focus. 
They  will  stand  out  as  clearly  defined  colored  objects  on  a  bright 
field. 


82  BINOCULAR  MICROSCOPES  [Cn.  Ill 

If  one  closes  the  diaphragm  until  i  or  J  of  the  aperture  of  the 
objective  is  used,  the  image  will  be  a  combined  color  and  refraction 
image. 

§  140.  Shading  the  object.  —  To  get  the  clearest  image  of  an  object 
no  light  should  reach  the  eye  except  from  the  object.  A  handkerchief 
or  a  dark  cloth  wound  around  the  objective  will  serve  the  purpose. 
Often  the  proper  effect  may  be  obtained  by  simply  shading  the  top 
of  the  stage  with  the  hand  or  with  a  piece  of  card-board.  Unless  one 
has  a  very  favorable  light  the  shading  of  the  object  is  of  the  greatest 
advantage,  especially  with  the  homogeneous  immersion  objectives. 
The  shield  (fig.  33)  is  the  most  satisfactory  means  for  this  purpose, 
as  the  entire  microscope  above  the  illuminating  apparatus  is  shaded. 
This  screen  also  shades  the  face  of  the  observer. 

§  141.  Cleaning  homogeneous  objectives.  —  After  one  is  through 
with  a  homogeneous  objective,  it  should  be  carefully  cleaned  as  fol- 
lows: Wipe  off  the  homogeneous  liquid  with  a  piece  of  the  lens  paper 
(§  158);  then  if  the  fluid  is  cedar  oil,  wet  one  corner  of  a  fresh  piece 
in  xylene,  or  chloroform,  and  wipe  the  front  lens  with  it.  Immediately 
afterward  wipe  with  a  dry  part  of  the  paper.  The  cover-glass  of  the 
preparation  can  be  cleaned  in  the  same  way.  If  the  homogeneous 
liquid  is  a  glycerin  mixture  proceed  as  above,  but  use  water  to 
remove  the  last  traces  of  glycerin. 

BINOCULAR  MICROSCOPES 

§  142.  For  a  binocular  arrangement  which  shall  be  equally  good 
for  all  powers  up  to  and  including  the  highest  oil  immersion,  the 
following  fundamental  requirements  must  be  met: 

1.  The  light  to  each  eye  should  be  of  equal  intensity. 

2.  The  optical  path  of  the  light  to  each  eye  should  be  of  the  same 
length,  so  that  the  magnification  of  the  two  images  will  be  the  same. 

3.  The  numerical  aperture  should  not  be  cut  down  or  disturbed  in 
any  way. 

4.  The  diffraction  effects  should  be  the  same  as  for  the  monocular 
microscope. 

5.  The  oculars  must  be  laterally  adjustable  for  the  pupillary  dis- 
tance of  different  observers. 


CH.  Ill]  BINOCULAR  MICROSCOPES  83 

6.  Pairs  of  oculars  of  different  powers  should  be  usable,  for  ex- 
ample a  pair  of  5x,  a  pair  of  rox  oculars,  etc.,  that  is,  the  oculars  should 
not  need  to  be  special  and  should  not  be  limited  to  one  set. 

7.  It  should  be  possible  to  focus  one  tube  independently  to  com- 
pensate for  difference  in  the  two  eyes  of  the  observer. 

8.  It  should  be  possible  to  focus  the  entire  microscope  with  one 
coarse  and  one  fine  adjustment  as  for  monocular  instruments. 

9.  The  tubes  may  converge  so  that  the  axes  of  the  eyes  will  be 
directed  to  the  near  point  of  vision  (250  mm.). 

10.  The  tubes  may  be  parallel,  then  the  axes  of  the  eyes  will  be 
parallel  as  for  looking  at  distant  objects. 

§  143.  Very  early  in  the  history  of  the  telescope  and  of  the  com- 
pound microscope,  as  nature  has  endowed  us  with  two  eyes,  it  was 
insisted  upon  that  both  eyes  should  be  used  in  examining  objects  in- 
stead of  using  only  one  eye.  This  required  two  similar  microscopes 
or  telescopes  side  by  side  and  the  right  distance  apart  for  the  two  eyes. 
There  still  persists  in  the  common  opera-glasses  the  original  binocular 
Dutch  telescope-microscope. 

The  modern  binocular  dissecting  microscope  with  two  tubes  and 
two  objectives  and  two  oculars  is  in  principle  like  the  original  binocular 
microscope  of  Cherubin  d'Orleans  (1677),  except,  of  course,  the  earlier 
one  had  no  erecting  arrangement. 

The  double  microscope  with  two  complete  tubes,  two  objectives, 
and  two  oculars  is  not  available  for  high  powers,  for  the  two  objectives 
cannot  be  close  enough  together  to  bring  the  exceedingly  small  object 
into  the  field  of  both  microscopes  at  the  same  time.  Naturally, 
therefore,  an  effort  was  made  to  use  a  single  objective  and  to  divide 
the  light  passing  through  it  so  that  half  should  go  to  the  right  and  half 
to  the  left  eye.  The  first  successful  binocular  of  this  kind  was  invented 
by  Riddell  of  New  Orleans  in  America  in  1851.  In  this,  four  prisms 
are  used  just  above  the  objective  and  serve  to  divide  the  light  equally 
and  to  pass  it  on  to  the  two  eyes  through  two  parallel  tubes,  each  with 
its  own  ocular.  Later  a  satisfactory  form  was  invented  by  Mr. 
Wenham  of  England  in  which  there  is  but  a  single  prism  (fig.  52). 
Neither  of  these  forms  permitted  of  very  high  powers.  Finally,  in 
1864,  Mr.  Robert  B.  Tolles  invented  a  binocular,  stereoscopic  eye- 


84 


BINOCULAR  MICROSCOPES 


[CH.  Ill 


piece  for  use  on  any  monocular  microscope.     The  prisms  divided  the 
light  equally  and  it  was  sent  up  through  tubes  parallel  with  the  main 


FIG.  52.     WENHAM'S  BINOCULAR  MICROSCOPE. 
(From  Carpenter). 

A  Section  of  the  microscope  with  the  two  converging  tubes.  By  pulling 
out  the  draw-tubes  the  oculars  are  separated  for  the  correct  pupillary  distance 
of  each  observer. 

L  R     The  axes  of  the  left  and  right  tubes. 
*  a     The  prism  which  divides  the  light  from  the  object. 

c  b     The  field  lenses  of  the  two  oculars. 

B     Enlargement  of  the  dividing  prism. 

a,  b,  c,  d     Path  of  the  light  in  the  prism  for  the  left  eye. 

As  shown,  the  light  to  the  right  eye  extends  straight  upward.  This  ar- 
rangement is  limited  to  rather  low  powers. 

tube  of  the  microscope,  but,  of  course,  separated  the  proper  distance 
for  the  two  eyes.  In  the  words  of  President  F.  P.  Barnard  of  Colum- 
bia College,  New  York,  "This  binocular  eye-piece  works  with  objec- 
tives of  all  powers  with  perfect  equality  of  illumination  in  both  fields." 


CH.  Ill] 


BINOCULAR  MICROSCOPES 


While  more  or  less  successful  efforts  had  long  been  made  to  produce 
binocular  microscopes  with  a  single  objective,  the  optical  requirements 


Ocular  1 


ocu/. 


FIG.  53.    IVES  BINOCULAR  ARRANGEMENT  FOR  ALL  POWERS. 
(Journal  of  the  Franklin  Institute,  Dec.  1902). 

Objective     The  single  objective. 

pb     The  prism  box  at  the  lower  end  of  the  tube. 

a,  b,  c     The  prisms  dividing  the  light  equally  from  each  point  to  the  two  eyes. 

o,  b  The  transparent  silvered  surface  in  the  prism  allowing  half  the  light  to 
pass  through  and  half  to  be  reflected  to  the  right. 

c  Prism  at  the  right  reflecting  the  light  upward  to  the  right  eye,  as,  ad- 
justing screw  to  tilt  the  prism  c,  at  the  correct  angle  for  the  position  of  the 
right  ocular. 

apd     Adjustment  for  the  pupillary  distance. 

Ocular,  /,  Ocular  2     The  oculars  for  the  right  and  the  left  eye. 

Axis  i     The  principal  optic  axis  for  the  left  eye. 

Axis  2     The  principal  optic  axis  for  the  right  eye. 

Due  to  the  length  of  the  prism  c,  this  axis  is  optically  of  the  same  length  as 
Axis  i  for  the  left  eye. 

were  not  fully  grasped.  Recently,  however,  Mr.  Frederic  E.  Ives 
has  stated  the  optical  principles  with  great  clearness,  and  shown  how 
binocular  microscopes  using  a  single  objective  can  be  constructed. 


86  BINOCULAR  MICROSCOPES  [Cn.  HI 

(Ives,  Jour.  Franklin  Institute,  Dec.  1902,  pp.  441-445,  fig.  53.) 
See  also  the  paper  by  Conrad  Beck,  with  figures  of  the  various 
forms  of  dividing  prisms  of  binoculars,  Jour.  Roy.  Micr.  Soc.,  1914, 
pp.  17-23  (fig.  54,  55). 

All  single-objective  binocular  microscopes  now  on  the  market  are 
made  in  accordance  with  the  principles  enunciated  in  Mr.  Ives' 
original  paper. 

§  144.  Parallel  or  converging  tubes  for  binoculars.  —  As  men- 
tioned above,  the  original  telescope-microscope,  persisting  in  the 
form  of  the  opera-glass,  had  parallel  tubes.  Following  the  differentia- 
tion of  the  telescope  and  microscope  in  which  the  objective  of  the  micro- 
scope gradually  became  of  smaller  diameter  and  shorter  focus,  and  the 
eye-piece  of  the  original,  concave  Dutch  form  was  replaced  by  the 
convex,  Keplerian  form  (see  history  at  the  end),  the  two  objectives 
of  the  binocular  could  be  placed  closer  together,  and  in  this  way  smaller 
and  smaller  objects  could  be  brought  into  the  same  field  even  with 
quite  high  objectives.  As  the  oculars  must  be  separated  sufficiently 
to  bring  their  axes  in  the  middle  of  the  pupil  of  the  eye,  the  tubes  must 
be  made  more  or  less  converging  (fig.  52). 

The  question  is,  which  arrangement,  parallel  or  converging  tubes, 
is  easiest  on  the  eyes  of  the  observer  for  continuous  work.  The  argu- 
ment of  those  advocating  the  parallel  arrangement  is  that  when  the 
eyes  are  at  rest,  as  in  viewing  distant  objects,  the  rays  entering  the 
eyes  are  practically  parallel,. and  the  two  eye  axes  are  of  course  also 
parallel;  and  that  with  the  most  favorable  focus  of  the  microscope 
the  rays  of  light  leaving  the  oculars  are  practically  parallel,  hence 
the  eyes  should  have  their  axes  parallel  as  for  viewing  distant  objects, 
and  that  there  is  no  effort  at  accommodation  for  getting  the  sharpest 
image  on  the  retina. 

For  those  who  advocate  converging  tubes  it  is  pointed  out  that  in 
observing  small  details  the  eye  naturally  uses  the  near  point  of  distinct 
vision,  viz.  250  mm.  for  adults,  and  not  the  far  point,  and  therefore 
the  most  satisfactory  microscopic  work  can  be  accomplished  with 
the  converging  tubes  to  correspond  with  the  natural  convergence 
of  the  eyes.  Much  actual  experience  will  doubtless  be  required  for 
the  settlement  of  the  question.  From  the  author's  experience  with  the 


CH.  Ill] 


BINOCULAR  MICROSCOPES 


monocular  microscope,  it  seems  that  the  argument  that  the  eye  should 
be  in  a  condition  of  rest  and  not  of  accommodation  when  doing  micro- 
scopic work,  that  is,  the  argument  for  the  parallel  tubes,  seems  con- 
vincing. Perhaps  the  Yankee  spirit  of  compromise  will  be  found  most 


FIG.  54-55. 


Object 


PRISM  ARRANGEMENT    FOR  Two 
ALL  POWERS. 


Object 

FORMS  OF  BINOCULARS  FOR 


(Conrad  Beck,  Jour.  Roy.  Micr.  Soc.  1914). 


In  fig.  54  the  arrangement  is  for  parallel  tubes,  and  in  fig.  55  for  converging 
tubes. 

Object     The  object. 

Ob     The  objective. 

I,  r',  l,r  The  right  and  left  beams  of  light  emanating  from  the  same  point 
of  the  object. 

As  these  beams  extend  through  the  objective  and  into  the  prisms  they  are 
equally  divided  so  that  half  the  right  beam  goes  to  the  left  and  half  to  the  right 
eye,  and  so  with  the  left  beam.  This  is  indicated  by  the  heavy  and  light 
broken  lines  by  which  the  two  beams  are  indicated. 

i,  2,  .j,  4;  i,  2  The  four  prisms  in  fig.  54,  and  the  two  prisms  in  fig.  55. 
The  prisms  are  of  the  necessary  length  to  make  the  optical  path  of  the  light 
equal  for  the  two  tubes,  hence  the  magnification  is  equal  for  the  two  eyes. 

truly  practical  in  this  matter  and  the  binocular  tubes  of  the  future 
will  be  neither  parallel  nor  too  convergent. 

§  145.  Dissecting  spectacles.  —  Various  devices  have  been  pro- 
duced from  time  to  time  to  connect  directly  in  some  way  with  the  eye. 
The  long-used  watchmaker's  or  jeweler's  eye  glass  is  the  most  familiar 
example,  and  answers  fairly  well,  although  for  most  dissecting  work  it 


BINOCULAR  MICROSCOPES  [CH.  Ill 

is  more  satisfactory  to  have  the  magnifier  held  by  some  mechanical 
means  like  the  focusing  holder  shown  in  fig.  19. 

The  advantage  of  using  both  eyes  has  led  to  the  production  of 
binocular  arrangements  to  be  held  in  place  by  a  band  around  the  head. 
These  are  necessarily  rather  expensive. 

The  needs  were  stated  to  Dr.  A.  C.  Durand  in  1913,  and  at  my 
request  he  devised  a  pair  of  spectacles  which  magnified  approximately 
1.5  diameters.  In  addition  to  the  magnifying  curve  he  added  the 
correction  for  astigmatism,  and  combined  these  corrections  and  curves 
with  a  4  degree  prism,  base  in,  to  "  relieve  the  excessive  convergence 
which  would  otherwise  be  necessary  with  such  short  focus  spheres." 
With  these  spectacles,  which  are  much  cheaper  than  any  device  on 
the  market,  and  which  have  all  the  corrections  needed  for  the  eyes  of 
the  individual  observer,  it  is  very  easy  to  carry  on  minute  dissection. 
The  prisms  serve  to  prevent  the  weariness  which  comes  so  soon  with 
great  convergence.  The  eyes  look  nearly  straight  ahead,  as  in  viewing 
distant  objects,  and  are  therefore  in  position  of  rest.  These  spectacles 
have  also  been  found  of  much  service  in  reading  proof  of  fine  print. 

EXPERIMENTS  WITH  BINOCULAR  MICROSCOPES 
§  146.  Erecting,  double-objective  binocular.  —  Put  a  pair  of  ob- 
jectives of  40  to  50  mm.  focus,  and  a  pair  of  oculars,  4x  or  5x,  in  place. 
The  oculars  are  put  in  place  in  the  ordinary  manner  (§45),  but  the 
objectives  are  now  often  mounted  in  a  sliding  objective  changer  per- 
manently. To  put  them  in  place  one  has  simply  to  slide  the  pair  in 
the  mounting  into  the  proper  groove  at  the  lower  end  of  the  micro- 
scope body. 

Place  the  microscope  where  a  good  light  can  be  had  and  put  on  the 
stage  some  transparent  specimen  like  an  organ  with  the  blood  vessels 
injected  or  with  both  the  blood  and  the  lymphatic  vessels  injected. 
Reflect  the  light  up  through  the  specimen,  and  focus. 

§  147.  Arranging  the  microscope  for  binocular  vision.  —  Until  one 
has  had  some  experience  with  binocular  microscopes  it  is  not  easy  to 
tell  whether  one  is  seeing  with  one  eye  or  with  both.  In  order  to  see 
with  both  eyes  it  is  of  course  necessary  that  each  eye  should  receive 
the  beam  of  light  from  its  own  ocular  at  the  same  time,  and  this  can 


CH.  Ill]  BINOCULAR  MICROSCOPES  89 

occur  only  when  the  oculars  are  spread  the  right  amount  to  bring  the 
eye-points  the  same  distance  apart  as  the  pupils  of  the  eyes  of  the 
observer,  and  the  eyes  are  at  the  correct  level. 

Hold  the  head  close  to  the  oculars  and  look  into  the  microscope. 
Focus  as  usual  and  the  image  will  be  satisfactory.  Now  to  tell  whether 
the  image  is  seen  with  one  eye  or  with  both,  hold  the  head  still  and  shut 
the  eyes  alternately.  If  only  one  eye  is  being  used  no  image  at  all 
will  be  seen  when  that  eye  is  closed,  but  when  the  other  is  closed  there 
will  be  no  change  in  the  appearance  (§  147  a). 

If  it  is  found  that  only  one  eye  is  being  used,  change  the  spread  of 
the  oculars  by  grasping  the  prism  holder  or  drums  or  the  tubes  above 
these  with  the  two  hands  and  increase  and  diminish  the  distance  be- 
tween the  tubes  until  both  eyes  are  receiving  the  light,  and  there  is 
an  image  in  each  eye.  When  this  occurs  and  one  once  gets  the  stereo- 
scopic effect  there  will  never  be  any  doubt  in  the  future  whether  the 
vision  is  monocular  or  binocular. 

§  147a.  In  some  makes  of  binocular  microscopes  (the  Spencer  Lens  Co. 's, 
for  example),  there  is  a  little  shutter  just  above  the  objectives  which  can  be 
turned  to  either  side,  covering  the  back  of  the  corresponding  objective.  If 
the  image  is  still  apparent  whichever  objective  is  covered  then  of  course  both 
eyes  are  seeing  the  image,  but  if  the  image  is  wholly  obliterated  when  the  shut- 
ter is  on  one  side,  that  is  the  only  side  giving  an  image,  and  the  tubes  must  be 
changed  in  position  to  get  the  correct  pupillary  distance  of  the  eye-points. 

§  148.  Focusing  if  the  eyes  are  unlike.  —  It  occasionally  happens 
that  the  eyes  of  the  observer  are  markedly  different.  Provision  is 
made  for  focusing  one  tube  or  one  objective  to  compensate  for  this. 
If  it  is  necessary  to  make  this  special  adjustment,  focus  first  with  the 
rack  and  pinion  and  get  the  focus  as  sharp  as  possible  for  the  tube 
having  no  special  adjustment;  then,  without  changing  the  general 
focus,  turn  the  milled  ring  of  the  other  tube  until  the  image  for  the 
corresponding  eye  is  also  perfectly  sharp.  If  now  one  uses  both  eyes 
the  images  should  be  equally  sharp  and  the  binocular  vision  good. 

Of  course  the  lower  the  objective  the  less  need  there  is  for  special 
adjustment. 

§  149.  Opaque  objects  for  the  double-objective  binocular.  —  Put 
a  piece  of  black  paper  or  velvet  on  the  stage,  and  upon  that  a  piece 


90  BINOCULAR  MICROSCOPES  [CH.  Ill 

of  white  cloth,  a  light-colored  insect,  a  feather,  or  any  other  object 
which  it  is  desired  to  see.  Place  the  microscope  where  there  is  a  good 
light  and  look  at  the  object.  When  seeing  with  both  eyes  the  stereo- 
scopic effect  will  be  very  striking,  and  one  can  see  the  different  levels, 
etc.,  as  with  the  naked  eye. 

For  dissecting  and  for  dark  objects  the  lighting  must  be  brilliant. 
Sunshine  on  the  specimen  is  often  none  too  strong,  but  as  that  is  not 
stationary  and  not  to  be  had  at  all  times,  it  is  usually  more  satisfactory 
to  use  a  small  arc  lamp  (fig.  49)  or  a  projector  with  a  concentrated  or 
stereopticon  type  mazda  lamp.  If  the  light  is  concentrated  upon 
the  mirror  for  translucent  specimens  or  directly  upon  the  opaque 
specimens  there  will  be  sufficient  light  to  give  satisfactory  images. 

§  149a.  Correct  movement  of  the  specimen  or  instruments  under  an 
erecting  microscope.  —  For  one  who  has  become  thoroughly  trained  in  using 
the  ordinary  inverting  compound  microscope  it  is  very  difficult  to  make  the 
proper  motions  to  move  the  specimen,  or  to  move  the  dissecting  instruments 
correctly  under  an  erecting  compound  microscope.  This  illustrates  the  power 
of  training.  The  beginner  with  the  inverting  microscope  finds  it  hard  to  move 
his  hands  in  the  opposite  way  from  what  his  eyes  dictate,  but  when  the  correla- 
tion between  the  appearance  and  the  motion  necessary  has  become  fixed,  it  is 
equally  difficult  to  move  the  hands  in  the  direction  which  the  eyes  indicate, 
although  it  is  known  that  this  is  now  correct.  This  difficulty  is  soon  over- 
come by  practice. 

Under  the  simple  microscope,  however,  in  which  there  is  no  reversal  or  in- 
version, the  eyes  and  hand  work  together  automatically  as  with  the  naked  eye. 

SINGLE-OBJECTIVE  BINOCULAR  MICROSCOPES  FOR  ALL  POWERS 

§  150.  Single-objective  binoculars.  —  This  is  to  be  used  for 
looking  at  microscopic  specimens  exactly  as  a  monocular  compound 
microscope.  That  is,  the  lighting,  numerical  aperture  of  the  condenser, 
and  all  the  work  done  with  it  is  the  same.  The  only  difference  is  that 
the  two  tubes  are  to  be  arranged  at  the  right  distance  apart  to  give 
binocular  vision  as  discussed  in  §  147. 

If  the  two  eyes  differ,  then  one  of  the  tubes  must  be  focused  to  make 
the  necessary  compensation. 

With  the  excellent  binoculars  now  available,  there  is  nothing  done 
with  the  monocular  microscope  that  cannot  be  done  with  the  binocular, 
and  for  many  workers  the  use  of  the  two  eyes,  as  has  been  so  long 
contended  by  the  English  microscopists,  gives  much  relief  for  long- 


CH.  Ill]  BINOCULAR  MICROSCOPES  91 

continued  observation.  The  stereoscopic  appearance,  while  desirable 
in  some  cases,  is  not  of  very  much  advantage  in  revealing  structure 
when  working  with  high  powers. 

§  151.  Experiments  with  single  objective  binoculars.  —  So  far 
as  the  lighting  is  concerned  it  is  exactly  as  for  a  monocular  microscope 
(§  83-130). 

§  152.  Arrange  the  microscope  in  a  convenient  position,  use  any 
pair  of  oculars  ($x  or  lox)  and  any  objective,  but  to  begin  with  a 
low-power  (16  mm.)  objective  is  to  be  preferred.  Use  a  preparation 
like  the  mounted  fly's  wing  or  a  preparation  showing  injected  blood 
vessels.  Look  into  the  microscope  and  arrange  the  light  so  that  the 
object  is  well  illuminated.  The  determination  of  binocular  vision 
is  exactly  as  with  the  double-objective  binocular  (§  147). 

§  153.  Pupillary  distance.  —  To  vary  the  distance  of  the  eye-points 
for  converging  tubes  rotate  the  oculars  equally  up  from  the  lowest 
point  until  the  binocular  effect  is  secured,  and  then  note  the  position 
of  the  oculars.  If  the  tubes  are  parallel  there  is  a  screw  between  them 
by  which  they  can  be  separated  or  approximated.  Continue  to  adjust 
until  the  binocular  vision  is  perfect  and  then  note  the  position  so 
that  the  tubes  can  be  set  in  the  right  position  instantly  at  some 
future  time.  Each  individual  must  determine  the  pupillary  distance 
of  his  own  eyes.  The  chances  are  against  any  two  persons  being  alike 
in  that  respect. 

§  154.  Unlikeness  of  the  two  eyes.  —  With  many  persons  the 
refraction  of  the  two  eyes  is  somewhat  different,  so  that  if  the  micro- 
scope is  in  focus  for  one  eye  it  is  necessary  to  refocus  for  the  other. 
Now  if  this  is  the  case  it  is  necessary  to  focus  the  two  tubes  differently 
in  a  binocular.  Focus  first  with  the  tube  which  is  not  adjustable  for 
parallel  tubes,  or  with  either  one  where  converging  tubes  are  used. 
Then  close  the  eye  used  for  focusing  first  and  focus  the  other  tube  for 
the  other  eye  by  rotating  the  tube  up  or  down  until  the  image  is  sharp, 
or  by  turning  the  milled  ring  in  the  adjusting  tube  of  the  parallel  tube 
type.  If  one  now  looks  into  the  microscope  with  both  eyes  there  will 
be  two  sharp  images  fused. 


92  CARE  OF  THE  MICROSCOPE  [Cn.  Ill 

CARE  OF  THE  MICROSCOPE 

§  155.  The  microscope  should  be  handled  carefully  and  kept  per- 
fectly clean.  The  oculars  and  objectives  should  never  be  allowed  to 
fall. 

When  not  in  use  keep  it  in  a  place  as  free  as  possible  from  dust. 

All  parts  of  the  microscope  should  be  kept  free  from  liquids,  espe- 
cially from  acids,  alkalies,  alcohol,  xylene,  turpentine,  and  chloroform. 

§  156.  Care  of  the  mechanical  parts.  —  To  clean  the  sliding  me- 
chanical parts  put  a  small  quantity  of  some  fine  oil  (olive  oil  or  liquid 
vaselin  and  gasoline  or  xylene,  equal  parts)  on  a  piece  of  gauze,  chamois 
leather,  or  lens  paper,  and  rub  the  parts  well ;  then  with  a  clean  dry 
piece  of  the  cloth  chamois  or  paper  wipe  off  most  of  the  oil.  If  the 
mechanical  parts  are  kept  clean  in  this  way  a  lubricator  is  rarely  needed. 
When  opposed  brass  surfaces  "cut,"  i.e.,  when  from  the  introduc- 
tion of  some  gritty  material,  minute  grooves  are  worn  in  the  opposing 
surfaces,  giving  a  harsh  movement,  the  opposing  parts  should  be 
separated,  carefully  cleaned  as  described  above,  and  any  ridges  or 
prominences  scraped  down  with  a  knife.  Where  the  tendency  to 
"cut"  is  marked,  a  very  slight  application  of  equal  parts  of  beeswax 
and  tallow,  well  melted  together,  serves  a  good  purpose.  The  thick 
fibrous  grease  such  as  is  used  in  the  grease  cups  of  automobiles  is  also 
good. 

In  cleaning  lacquered  parts,  xylene  alone  answers  well,  but  it  should 
be  quickly  wiped  off  with  a  clean  piece  of  the  lens  paper.  Do  not  use 
alcohol,  as  it  dissolves  the  lacquer. 

§  157.  Care  of  the  optical  parts.  —  These  must  be  kept  scrupu- 
lously clean  in  order  that  the  best  results  may  be  obtained. 

Glass  surfaces  should  never  be  touched  with  the  fingers,  for  that 
will  soil  them. 

Whenever  an  objective  is  left  in  position  on  a  microscope,  or  when 
several  are  attached  by  means  of  a  revolving  nose-piece,  an  ocular 
should  be  left  in  the  upper  end  of  the  tube  to  prevent  dust  from  fall- 
ing down  upon  the  back  lens  of  the  objective  (§  i$7a). 

§  157a.  As  pointed  out  by  Wright,  p.  93,  one  of  the  surest  ways  to  detect 
anything  wrong  with  the  objective  is  to  examine  the  eye-point  with  a  magnifier. 


CH.  Ill]  CARE  OF  THE  MICROSCOPE  93 

The  field  should  be  lighted  well  and  the  aperture  of  the  objective  filled  about 
f  full  of  light.  If  there  are  any  defects  as  smears  of  balsam  or  liquids  on 
the  front  lens,  unsealing  of  the  combinations,  or  dust  on  the  upper  face  of  the 
back  lens  the  defect  can  be  seen  in  the  eye-point. 

Another  and  very  certain  method  of  detecting  imperfections  is  to  rotate  the 
different  elements  while  looking  into  the  microscope.  If  the  defect  is  in  the 
mirror  they  will  change  in  position  when  the  mirror  is  moved,  and  so  with  all 
the  other  elements.  Defects  in  the  ocular  are  strikingly  shown  by  rotating  it. 

§  158.  Lens  paper.  —  The  so-called  Japanese  filter  paper,  which, 
from  its  use  with  the  microscope,  I  have  designated  lens  paper,  has 
been  used  in  the  author's  laboratory  since  1884  for  cleaning  the  lenses 
of  oculars  and  objectives,  and  especially  for  removing  the  fluid  used 
with  immersion  objectives.  Whenever  a  piece  is  used  once  it  is  thrown 
away.  It  has  proved  more  satisfactory  than  cloth  or  chamois,  be- 
cause dust  or  sand  is  not  present;  and  from  its  bibulous  character  it 
is  very  efficient  in  removing  liquid  or  semi-liquid  substances. 

§  159.  Removal  of  dust,  etc.  —  (i)  Dust  may  be  removed  with 
a  camel's  hair  brush,  or  by  wiping  with  the  lens  paper. 

(2)  Cloudiness  may  be  removed  from  the  glass  surfaces  by  breath- 
ing on  them,  then  wiping  quickly  with  a  soft   cloth  or  the  lens 
paper. 

Cloudiness  on  the  inner  surfaces  of  the  ocular  lenses  may  be  removed 
by  unscrewing  them  and  wiping  as  directed  above.  A  high  objective 
should  never  be  taken  apart  by  an  inexperienced  person. 

If  the  cloudiness  cannot  be  removed  as  directed  above,  moisten 
one  corner  of  the  cloth  or  paper  with  95%  alcohol,  wipe  the  glass  first 
with  this,  then  with  the  dry  cloth  or  the  paper. 

(3)  Water  may  be  removed  with  soft  cloth  or  the  lens  paper. 

(4)  Glycerin  may  be  removed  with  cloth  or  lens  paper  saturated 
with  distilled  water;  remove  the  water  as  above. 

(5)  Blood  or  other  albuminous  material  may  be  removed  while 
fresh,  the  same  as  glycerin.     If  the  material  has  dried  on  the  glass,  it 
may  be  removed  more  readily  by  adding  a  small  quantity  of  ammonia 
to  the  water  in  which  the  cloth  is  moistened  (water  100  c.c.,  ammonia 

I  C.C.). 

(6)  Canada  Balsam,  damar,  paraffin,  or  any  oily  substance  may 
be  removed  with  a  cloth  or  paper  wet  with  chloroform,  gasoline  or 
xylene.     The  application  of  these  liquids  and  their  removal  with  a 


94  CARE  OF  THE  EYES  [Cn.  Ill 

soft  dry  cloth  or  lens  paper  should  be  as  rapid  as  possible,  so  that  none 
of  the  liquid  will  have  time  to  soften  the  setting  of  the  lenses. 

(7)  Shellac  Cement  may  be  removed  by  the  paper  or  a  cloth  moist- 
ened in  95%  alcohol. 

(8)  Brunswick  Black,  Gold  Size,  and  all  other  substances  soluble 
in  chloroform,  etc.,  may  be  removed  as  directed  for  balsam  and  damar. 

In  general,  use  a  solvent  of  the  substance  on  the  glass  and  wipe  it 
off  quickly  with  a  fresh  piece  of  the  cloth  or  lens  paper. 

It  frequently  happens  that  the  upper  surface  of  the  back  combina- 
tion of  the  objective  become  dusty.  This  may  be  removed  in  part 
by  a  brush,  but  more  satisfactorily  by  using  a  piece  of  the  lens  paper 
loosely  twisted.  When  most  of  the  dust  is  removed  some  of  the  paper 
may  be  put  over  the  end  of  a  pine  stick  (like  a  match  stick)  and  the 
glass  surfaces  carefully  wiped. 

CARE  OF  THE  EYES 

§  160.  Keep  both  eyes  open,  using  the  eye-shade  if  necessary  (fig. 
56),  and  divide  the  labor  between  the  two  eyes,  using  one  eye  for  a 


FIG.  56.     EYE-SHADE  FOR  THE  TOP  OF  THE    MICROSCOPE    TO  ENABLE  THE 
OBSERVER  TO  KEEP  BOTH  EYES  OPEN. 

while  and  then  the  other.  It  frequently  happens  that  one  eye  is  much 
more  perfect  than  the  other,  then  of  course  the  more  perfect  eye  is 
used  all  the  time. 


CH.  Ill] 


CARE  OF  THE  EYES;    WORK  TABLE 


95 


The  binocular  microscope  has  certain  advantages  in  that  one  uses 
both  eyes  all  the  time  as  in  naked-eye  observation.  If  a  binocular  is 
used,  however,  one  must  adjust  it  accurately  so  that  each  eye  sees 
an  equally  sharp  image  (§  154). 

§  161.  In  the  beginning  it  is  not  advisable  to  look  into  the  micro- 
scope continuously  for  more  than  half  an  hour  at  a  time.  One  never 


FIG.  57.     LABORATORY  TABLE  AND  ADJUSTABLE  STOOL. 

This  table  is  122  cm.  long,  61  cm.  wide,  and  73  cm.  high  (2X4  feet  on  top,  and 
29  inches  high). 

The  corners  and  edges  are  rounded  and  the  top  is  stained  with  aniline  black. 
The  front  of  the  rail  is  cut  out,  and  the  drawer  is  at  the  right  so  that  it  can  be 
opened  without  moving  the  stool. 

should  work  with  the  microscope  after  the  eyes  feel  fatigued.  After 
one  becomes  accustomed  to  microscopic  observation  he  can  work  for 
several  hours  with  the  microscope  without  fatiguing  the  eyes.  This 
is  due  to  the  fact  that  the  eyes  become  inured  to  labor  like  the  other 
organs  of  the  body  by  judicious  exercise.  It  is  also  due  to  the  fact 
that  but  very  slight  accommodation  is  required  of  the  eyes,  the  eyes 


CARE    OF    THE    EYES;  WORK    TABLE 


[CH.  Ill 


remaining  nearly  in  a  condition  of  rest  as  for  distant  objects.  The 
fatigue  incident  upon  using  the  microscope  at  first  is  due  partly  at 
least  to  the  constant  effort  on  the  part  of  the  observer  to  remedy  the 
defects  of  focusing  the  microscope  by  accommodation  of  the  eyes. 
This  should  be  avoided  and  the  fine  adjustment  of  the  microscope 

used  instead  of  the  muscles 
of  accommodation.  With 
a  microscope  of  the  best 
quality,  and  suitable  light 
-  that  is,  light  which  is 
steady  and  not  so  bright 
as  to  dazzle  the  eyes  nor 
so  dim  as  to  strain  them 
in  determining  details  — 
microscopic  work  should 
improve  rather  than  injure 
the  sight. 

If   artifidal   H§ht    mUSt 


F,c.  S8.    M.CROSCOPICAL  LABORATORY  DESK 


be  used,  give  it  daylight 


WITH  MICROSCOPE  AND  CHALET  LAMP. 

(Desk  designed  by  Dr.  V.  A.  Moore    about  one    qualities  by  placing  a  piece 
twentieth  natural  size.)  of    ground    daylight   glass 

The  size  of  the  top  and  the  height  are  the  between  the  source  of  light 
same  as  for  the  laboratory  table  (fig.  57,  §162). 

m  At  the  right  there  is  a  cabinet  with  com-  and  the  microscope.     This 

bination  lock  (10,  d)  for  a  microscope,  and  w;ii  p.jve  one  a  verv  soft 
above  a  drawer  with  combination  lock  (a,d). 

At  the  right  is  a  writing  shelf  (5)  and  four  hght  like  that  from  a  white 

drawers  (b,  c,  d,  e.}  rlond  (S  02") 

Near  the  bottom  is  a  brace  (br)  which  also 

serves  as  a  foot  rest.  §162.     Position   and 

M    Compound  microscope  with   the   Chalet  character     of    the     work- 
Microscope  Lamp  in  front  of  it. 

table.  --  The    work-table 

should  be  very  firm  and  large  (61  X  122  cm.  on  top,  and  73  cm. 
high;  24  x  48  X  29  in.,  fig.  57),  so  that  the  necessary  apparatus 
and  material  for  work  may  not  be  too  crowded.  The  table  should 
also  be  of  the  right  height  to  make  work  by  it  comfortable.  An  ad- 
justable stool,  something  like  a  piano  stool,  is  convenient;  then  one 
may  vary  the  height  corresponding  to  the  necessities  of  special 
cases.  It  is  a  great  advantage  to  sit  facing  the  window  if  daylight 
is  used;  then  the  hands  do  not  constantly  interfere  with  the  il- 


CH.  Ill]  TESTING  THE  MICROSCOPE  97 

lumination.  To  avoid  the  discomfort  of  facing  the  light  a  shield  (fig. 
33)  is  very  useful.  For  advanced  students  and  private  workers  a  desk 
like  that  shown  in  fig.  58  is  very  convenient. 

TESTING  THE  MICROSCOPE 

§  163.  Testing  the  microscope.  —  To  be  of  real  value  this  must 
be  accomplished  by  a  person  with  both  theoretical  and  practical 
knowledge,  and  also  with  an  unprejudiced  mind.  Such  a  person  is 
not  common,  and  when  found  does  not  show  overanxiety  to  pass 
judgment.  Those  most  ready  to  offer  advice  should  as  a  rule  be 
avoided,  for  in  most  cases  they  simply  "have  an  ax  to  grind,"  and 
are  sure  to  commend  only  those  instruments  that  conform  to  the  "fad " 
of  the  day.  From  the  writer's  experience  it  seems  safe  to  say  that  the 
inexperienced  can  do  no  better  than  to  state  clearly  what  he  wishes 
to  do  with  a  microscope  and  then  trust  to  the  judgment  of  one  of  the 
optical  companies.  The  makers  of  microscopes  and  objectives  guard 
with  jealous  care  the  excellence  of  both  the  mechanical  and  optical 
part  of  their  work,  and  send  out  only  instruments  that  have  been  care- 
fully tested  and  found  to  conform  to  the  standard.  This  would  be 
done  as  a  matter  of  business  prudence  on  their  part,  but  it  is  believed 
by  the  writer  that  microscope  makers  are  artists  first  and  take  an 
artist's  pride  in  their  work;  they  therefore  have  a  stimulus  to  excel- 
lence greater  than  business  prudence  alone  could  give. 

What  has  just  been  said  does  not  by  any  means  imply  that  the 
purchaser  of  a  microscope  should  blindly  accept  anything  which  is 
offered  him.  It  simply  means  that  if  one  has  no  knowledge  of  a 
microscope  one  can  hardly  pass  expert  judgment  upon  it. 

§  164.  Mechanical  parts.  —  All  of  the  parts  should  be  firm,  and 
not  too  easily  shaken.  Bearings  should  work  smoothly.  The  mirror 
should  remain  in  any  position  in  which  it  is  placed  (fig.  25). 

§  165.  Focusing  adjustments.  —  The  coarse  or  rapid  adjustment 
should  be  by  rack  and  pinion  and  work  so  smoothly  that  even  the 
highest  power  can  be  easily  focused  with  it  by  an  experienced  observer. 

This  coarse  adjustment  is  liable  to  work  too  hard  or  too  easily.  If 
it  works  too  hard,  the  bearings  of  the  pinion  are  too  tight  or  the  gliding 
surfaces  are  sticky  and  not  properly  lubricated.  If  the  bearings  are 


98  TESTING  THE  MICROSCOPE  CCn.  Ill 

too  tight,  loosen  the  screws  very  slightly;  if  the  bearings  are  not  lubri- 
cated or  the  surfaces  are  covered  with  sticky  oil,  wet  a  cloth  with  a 
good  lubricating  oil  and  rub  the  gliding  surfaces  well.  This  will  clean 
them  and  lubricate  them  at  the  same  time. 

If  the  tube  runs  down  too  easily  the  bearings  of  the  pinion  are  too 
loose  and  the  screws  should  be  tightened  a  little. 

§  166.  The  fine  adjustment  is  more  difficult  to  deal  with.  —  From 
the  nature  of  its  purpose,  unless  it  is  approximately  perfect,  it  would 
be  better  off  the  microscope  entirely.  It  has  been  much  improved 
recently. 

It  should  work  smoothly  and  be  so  balanced  that  one  cannot  tell 
by  the  feeling  when  using  it  whether  the  screw  is  going  up  or  down. 
Then  there  should  be  absolutely  no  motion  except  in  the  direction  of 
the  optic  axis;  otherwise  the  image  will  appear  to  sway  even  with 
central  light.  Compare  the  appearance  when  using  the  coarse  and 
when  using  the  fine  adjustment.  There  should  be  no  swaying  of  the 
image  with  either  if  the  light  is  central  (§  116). 

§  167.  Testing  the  optical  parts.  —  As  stated  in  the  beginning, 
this  can  be  done  satisfactorily  only  by  an  expert  judge.  It  would  be 
of  very  great  advantage  to  the  student  if  he  could  have  the  help  of 
such  a  person.  In  no  case  is  a  microscope  to  be  condemned  by  an 
inexperienced  person.  If  the  beginner  will  bear  in  mind  that  his 
failures  are  due  mostly  to  his  own  lack  of  knowledge  and  lack  of  skill, 
and  will  truly  endeavor  to  learn  and  apply  the  principles  laid  down 
in  this  and  in  the  standard  works  referred  to,  he  will  learn  after  a 
while  to  estimate  at  their  true  value  all  the  parts  of  his  microscope. 

If  one  can  compare  a  new  or  unfamiliar  microscope  with  one  with 
which  there  is  entire  familiarity,  a  very  good  estimate  can  be  made. 
The  first  principle  is  to  use  some  microscope  with  which  one  is  familiar 
and  to  use  microscopic  preparations  of  which  one  knows  the  structure; 
then  a  fair  judgment  can  be  made  of  the  excellence  of  the  performance 
of  the  new  instrument.  If  there  seems  to  be  any  defect  in  the  image, 
make  sure 

(1)  that  the  lighting  is  good; 

(2)  that  the  proper  aperture  of  the  objective  is  being  used  and  that 
the  condenser  is  centered  (§  104); 


CH.  Ill]  CHOICE  OF  A  MICROSCOPE  99 

(3)  that  the  stage  is  shaded; 

(4)  that  the  tube-length  of  the  microscope  is  that  for  which  the 
objectives  were  corrected  (Ch.  IX). 

(5)  that  the  preparation  is  clean  and  gives  a  good  image  with  the 
microscope  with  which  one  is  familiar.     If  all  the  precautions  have 
been  taken  and  still  a  good  image  cannot  be  obtained  one  should  get 
some  more  expert  friend  or  the  makers  to  show  wherein  the  trouble 
lies. 

LABORATORY  AND  HIGH  SCHOOL  COMPOUND  MICROSCOPES 

§  168.  Optical  parts.  —  A  great  deal  of  beginning  work  with  the 
microscope  in  biological  laboratories  is  done  with  simple  and  inex- 
pensive apparatus.  Indeed  if  one  contemplates  the  large  classes  in 
the  high  schools,  the  universities,  and  medical  schools,  it  can  readily 
be  understood  that  microscopes  costing  from  $25  to  $50  each,  and  mag- 
nifying from  25  to  500  diameters,  are  all  that  can  be  expected.  But 
for  the  purpose  of  modern  histological  investigation  and  of  advanced 
microscopical  work  in  general,  a  microscope  should  have  something 
like  the  following  character:  Its  optical  outfit  should  comprise 
dry  objectives  of  50  mm.,  16-18  mm.  and  4  mm.  equivalent  focus. 
There  should  be  present  also  a  2  mm.  or  1.5  mm.  homogeneous  immer- 
sion objective.  Of  oculars  there  should  be  several  of  different  power. 

Even  in  case  all  the  optical  parts  cannot  be  obtained  in  the  begin- 
ning, it  is  wise  to  secure  a  stand  upon  which  all  may  be  used  when 
they  are  finally  secured. 

§  169.  Objectives.  —  Achromatic  objectives  will  serve  all  ordinary 
purposes.  For  photo-micrography  and  the  finest  work  where  the 
color  values  are  of  essential  importance,  the  apochromatic  objectives 
and  compensation  oculars  should  be  obtained,  if  possible,  although 
even  in  photography  and  the  most  difficult  fields  of  microscopy  the 
modern  achromatic  objectives  give  excellent  results. 

§  170.  Mechanical  parts  or  stand.  —  The  stand  should  be  low 
enough  so  that  it  can  be  used  in  a  vertical  position  on  an  ordinary 
table  without  inconvenience  (fig.  25,  38);  it  should  have  a  jointed 
(flexible)  pillar  for  inclination  at  any  angle  to  the  horizontal.  The 
adjustments  for  focusing  should  be  two,  —  a  coarse  adjustment  or 


ioo  CHOICE  OF  A  MICROSCOPE  [CH.  Ill 

rapid  movement  with  rack  and  pinion,  and  a  fine  adjustment  by  means 
of  a  micrometer  screw.  Both  adjustments  should  move  the  entire 
tube  of  the  microscope.  The  body  or  tube  should  be  short  enough 
for  objectives  corrected  for  the  short  or  160  millimeter  tube-length. 
It  is  an  advantage  to  have  the  draw- tube  graduated  in  centimeters 
and  millimeters.  The  lower  end  of  the  draw-tube  and  of  the  tube 
should  each  possess  a  standard  screw  for  objectives  (fig.  64).  The 
stage  should  be  quite  large  for  the  examination  of  slides  with  serial 
sections  and  other  large  objects.  The  substage  fittings  should  be 
so  arranged  as  to  enable  one  to  use  the  condenser  or  to  dispense 
entirely  with  it.  The  condenser  mounting  should  allow  up  and 
down  motion  (fig.  25). 

§  171.  Quality  and  cost.  —  Laboratory  microscopes  which  will 
answer  nearly  all  the  requirements  for  work  in  Biology,  including  His- 
tology, Embryology,  Pathology,  and  Bacteriology,  are  listed  in  the 
makers'  catalogues  at  about  $65~$75.  The  less  expensive  microscopes 
are  listed  at  $25-145.  Fortunately  in  the  State  of  New  York  the 
State  pays  half  for  high  school  apparatus,  so  that  there  is  no  reason 
why  each  high  school  should  not  be  properly  equipped  with  micro- 
scopes of  good  grade.  To  avoid  misunderstanding  it  should  be 
added  that  the  quality  of  the  oculars  and  objectives  on  the  cheaper 
microscopes  is  the  same  as  for  the  best  laboratory  microscopes. 
The  mechanical  work  also  is  of  excellent  quality. 

During  the  last  few  years  great  vigor  has  been  shown  in  the  micro- 
scopical world.  This  has  been  stimulated  largely  by  the  activity  in 
biological  and  chemico-physical'  science  and  the  widespread  appre- 
ciation of  the  microscope,  not  only  as  a  desirable,  but  as  a  necessary 
instrument  for  study  and  research.  The  production  of  the  new  kinds 
of  glass  and  the  apochromatic  objectives  has  been  a  no  less  potent 
factor  in  promoting  progress. 

MARKERS  AND  MECHANICAL  STAGES 

Markers  are  devices  to  facilitate  the  finding  of  some  object  or  part 
which  it  is  especially  desired  to  refer  to  again  or  to  demonstrate  to 
a  class.  The  mechanical  stage  makes  it  much  easier  to  follow  out  a 
series  of  objects  to  move  the  slide  when  using  high  powers,  and  for 


CH.  Ill] 


MARKERS  AND   MECHANICAL  STAGES 


101 


complete  exploration  of  a  preparation  and  for  blood  counting.  Most 
of  the  mechanical  stages  have  scales  and  verniers  by  which  an  object 
once  recorded  may  be  readily  found  again. 

§172.  Marker  for  preparations  (fig.  59). —This  instrument 
consists  of  an  objective-like  attachment  which  may  be  screwed  into 
the  nose-piece  of  the  microscope.  It  bears  on  its  lower  end  a  small 


59  60 

FIG.  59-60.     MARKERS  FOR  MICROSCOPICAL  SPECIMENS. 

FIG.  59.  The  simplest  form  of  marker.  It  consists  of  the  part  SS  with 
the  milled  edge  (M).  This  part  bears  the  society  or  objective  screw  for  at- 
taching the  marker  to  the  microscope.  R  Rotating  part  of  the  marker. 
This  bears  the  eccentric  brush  (B)  at  its  lower  end.  The  brush  is  on  the  wire 
(W).  This  wire  is  eccentric,  and  may  be  made  more  or  less  so  by  bending  the 
wire.  The  central  dotted  line  coincides  with  the  axis  of  the  microscope. 
The  revolving  part  is  connected  with  the  " Society  Screw"  by  the  small 
screw  (S). 

FIG.  60.  SS,  R,  and  B.  All  parts  same  as  with  fig.  59,  except  that  the 
brush  is  carried  by  a  sliding  cylinder,  the  end  view  being  indicated  in  B. 

brush  and  the  brush  can  be  made  more  or  less  eccentric  and  can  be 
rotated,  thus  making  a  larger  or  smaller  circle.  In  using  the  marker 
the  brush  is  dipped  in  colored  shellac  or  other  cement  and  when 
the  part  of  the  preparation  to  be  marked  is  found  and  put  exactly 
in  the  middle  of  the  field  the  objective  is  turned  aside  and  the  marker 
turned  into  position.  The  brush  is  brought  carefully  in  contact  with 
the  cover-glass  and  rotated.  This  will  make  a  delicate  ring  of  the 
colored  cement  around  the  object  (fig.  61).  Within  this  very  small 


IO2 


MARKERS   AND   MECHANICAL   STAGES 


[CH.  Ill 


9  © 


FIG.  61.  A  MICROSCOPICAL  SPECI- 
MEN WITH  A  SMALL  RING  EN- 
CLOSING THE  PART  OF  SPECIAL 
INTEREST. 


area  the  desired  object  can  be  easily  found  on  any  microscope.  The 
Jbrush  of  the  marker  should  be  cleaned  with  95%  alcohol  after  it  is 
used.  (Proc.  Amer.  Micro.  Soc.,  1894,  pp.  112-118). 

§  173.  Pointer  in  the  ocular.  —This 
is  a  slender  rod  of  some  sort  situated 
at  the  level  of  the  real  image  in  the 
microscope,  and  it  appears  with  the 
specimen  in  the  field  of  view. 

A  pointer  may  be  inserted  in  any 
ocular  as  follows: 

Remove  the  eye-lens  and  with  a 
little  mucilage  or  Canada  Balsam 

fasten  a  hair  from  a  camel's  hair  or  other  fine  brush  to  the  upper  sur- 
face of  the  diaphragm  (fig.  23-24)  so  that  it  will  project  about  half- 
way across  the  opening.  If  one  uses  this  ocular,  the  pointer  will 
appear  in  the  field  and  one  can  place 
the  specimen  so  that  the  pointer  in- 
dicates it  exactly,  as  in  using  a  pointer 
on  a  diagram  or  on  the  blackboard.  It 
is  not  known  to  the  author  who  de- 
vised this  method.  It  is  certainly  of 
the  greatest  advantage  in  demonstrat- 
ing objects  like  amcebas  or  white  blood 
corpuscles  to  persons  not  familiar  with 
them,  as  the  field  is  liable  to  have  in 
it  many  other  objects  which  are  more 
easily  seen. 

§  174.  Mechanical  stage.  —  For 
high  school  and  ordinary  laboratory 
work  a  mechanical  stage  is  not  needed ; 
but  for  much  work,  especially  where 
high  objectives  are  used,  a  mechanical 
stage  is  of  great  advantage.  It  is  also  advantageous  if  the  mechanical 
stage  can  easily  be  removed. 

The  one  found  on  the  most  expensive  American  and  English  micro- 
scopes for  the  last  twenty  years  and  the  one  now  present  on  the  larger 


FIG.   62-63.     POINTER    OCULAR 
AND  MICROSCOPIC  FIELD. 

P  P  The  pointer  attached  to 
the  diaphragm  of  the  ocular  and 
extending  out  into  the  free  space 
in  fig.  62.  In  fig.  63  the  pointer 
is  shown  indicating  the  position 
of  a  leucocyte. 


CH.  Ill]       ROYAL  MICROSCOPICAL  SOCIETY  STANDARDS 


103 


continental  microscopes  is  excellent  for  high  powers  and  preparations 
of  moderate  dimensions,  but  for  the  study  of  serial  sections  and  large 
sections  and  preparations  in  general,  a  form  of  mechanical  stage  which 
gives  great  lateral,  forward,  and  backward  movement,  and  which  is 
easily  removable,  is  desirable.  Such  removable  mechanical  stages 
are  now  produced  by  all  the  microscope  manufacturers.  The  latest 
and  best  forms  enable  one  to  explore  the  serial 
sections  on  slides  from  25  x  75  to  50  x  75  mm. 

ROYAL  MICROSCOPICAL  SOCIETY  STANDARDS 

§  175.  Society  screw.  —  Owing  to  the  lack 
of  uniformity  in  screws  for  microscope  objec- 
tives, the  Royal  Microscopical  Society  of 
London,  in  1857,  made  an  earnest  effort  to  in- 
troduce a  standard  size. 

In  order  to  facilitate  the  introduction  of 
this  universal  screw,  or,  as  it  soon  came  to  be 
called,  "The  Society  Screw,"  the  Royal  Micro- 
scopical Society  undertook  to  supply  standard 
taps.  From  the  mechanical  difficulty  in  mak- 
ing these  taps  perfect  there  soon  came  to 
be  considerable  difference  in  the  "  Society 
Screws,"  and  the  object  of  the  society  in  pro- 
viding a  universal  screw  was  partly  defeated. 
(See  Edward  Bausch,  Trans.  Amer.  Micr.  Soc.,  1884,  p.  153.) 

In  1884  the  American  Microscopical  Society  appointed  Mr.  Edward 
Bausch  and  Prof.  William  A.  Rogers  upon  a  committee  to  correspond 
with  the  Royal  Microscopical  Society,  with  a  view  to  perfecting  the 
standard  "  Society  Screw,"  or  of  adopting  another  standard  and  of 
perfecting  methods  by  which  the  screws  of  all  makers  might  be  truly 
uniform.  Although  this  matter  was  earnestly  considered  at  the  time 
by  the  Royal  Microscopical  Society,  the  mechanical  difficulties  were 
so  great  that  the  improvements  were  abandoned. 

Fortunately,  however,  during  the  year  1896  that  society  again  took 
hold  of  the  matter  in  earnest  and  the  "  Society  Screw  "  is  now  accu- 
rate, and  facilities  for  obtaining  the  standard  are  so  good  that  there 


FIG.  64.  ROYAL  MIC- 
ROSCOPICAL •  SOCIETY'S 
STANDARD  SCREW  FOR 
OBJECTIVES. 

(From  the  Jour.  Roy. 
Micr.  Soc.,  Aug.  1896). 


104  ROYAL  MICROSCOPICAL  SOCIETY  STANDARDS      [Cn.  Ill 

is  a  reasonable  certainty  that  the  universal  screw  for  microscopic 
objectives  may  be  realized.  It  is  astonishing  to  see  how  widely  the 
"  Society  Screw  "  has  been  adopted.  Indeed  there  is  not  a  maker 
of  first-class  microscopes  in  the  world  who  does  not  supply  the  objec- 
tives and  stands  with  the  "  Society  Screw,"  and  an  objective  in  Eng- 
land or  America  which  does  not  have  this  screw  should  be  looked  upon 
with  suspicion.  That  is,  it  is  either  old,  cheap,  or  not  the  product 
of  one  of  the  great  opticians.  For  the  Standard,  or  "  Society  Screw," 
see:  Trans.  Roy.  Micr.  Soc.,  1857,  pp.  30-41;  1859,  pp.  92-97;  1860, 
pp.  103-104.  (All  to  be  found  in  Quar.  Jour.  Micr.  Sci.,  o.  s., 
vols.  VI,  VII,  VIII).  Proc.  Amer.  Micr.  Soc.,  1884,  p.  274;  1886, 
p.  199;  1893,  p.  38.  Journal  of  the  Royal  Microscopical  Society, 
August,  1896. 

In  this  last  paper  of  four  pages  the  matter  is  very  carefully 
gone  over  and  full  specifications  of  the  new  screw  given.  It  con- 
forms almost  exactly  with  the  original  standard  adopted  by  the 
society,  but  means  have  been  devised  by  which  it  may  be  kept 
standard. 

The  following  discussion  and  specifications  are  from  the  Journal 
of  the  Royal  Microscopical  Society,  1915,  pp.  230-23 1.. 

"OBJECTIVE  SCREW  THREAD 

"  The  question  of  standardization  of  the  Objective  Screw  Thread 
was  first  discussed  by  the  Microscopical  Society  in  1857,  and  the  first 
sizing  tools  were  issued  in  1858. 

"  In  1896  the  Council  of  the  Royal  Microscopical  Society  issued 
another  Report,  and  drew  up  a  specification  defining  the  limits  of 
variation  allowable  from  the  original  standard  screw  thread. 

"  Difficulties  having  arisen  in  connexion  with  the  testing  and  ad- 
justing of  the  sizing  tools  supplied  by  the  Society,  the  Council  in  1911 
appointed  a  Gauges  Committee  to  look  into  the  question  of  obtain- 
ing and  testing  further  tools,  and  they  now  have  pleasure  in  informing 
Fellows  of  the  Society  that  an  arrangement  has  been  made  with  the 
Director  of  the  National  Physical  Laboratory  whereby  the  standard 
gauges  of  the  Society  have  been  deposited  at  the  National  Physical 
Laboratory.  The  Council  has  also  arranged  for  the  issue  of  new 


CH.  Ill]     ROYAL  MICROSCOPICAL  SOCIETY  STANDARDS  105 

objective  screw  sizing  taps  and  dies,  which  have  been  tested  and 
passed  by  the  N.P.L.  and  are  within  the  following  limits: 

"  Tap  for  sizing  Nose-pieces:  full  diameter  between  0.800  in. 
(=  20.3198  mm.)  and  0.803  m-  (=  20.3960  mm.). 

"  Die  for  sizing  Objective:  core  diameter  of  thread  between  0.7596 
in.  (=  19.2937  mm.)  and  0.7626  in.  (=  19.3699  mm.). 

"  A  certificate  of  accuracy  is  issued  with  each  tap  and  die.  These 
sizing  tools  are  now  on  sale,  and  may  be  obtained  by  application  to 
the  Secretaries  of  the  Royal  Microscopical  Society. 

"  The  standard  specification  for  the  objective  thread  has  not  been 
altered,  and  is  as  follows: 

"SPECIFICATION  OF  THE  ROYAL  MICROSCOPICAL  SOCIETY 
STANDARD  SCREW  THREAD  FOR  OBJECTIVES 

"  Metrical  Measurements  in  Brackets 

"Diameter. — 0.800  in.  [20.3198  mm.]. 

"  Pitch.  —  36  to  the  inch  [14.17  to  the  cm.]. 

"  Form.  —  Whitworth  screw,  i.e.  a  V-shaped  thread,  sides  of  thread 
inclined  at  an  angle  of  55°  to  each  other,  one-sixth  of  the  V  depth 
being  rounded  off  at  the  top  and  the  bottom  of  the  thread  (fig.  64). 

"  Length  of  Thread  on  Objective,  0.125  in.  (=  3.1750  mm.). 

"  Plain  Fitting  above  Thread  of  Objective,  o.i  in.  (=  2.5400  mm.) 
long,  not  to  exceed  0.759  m-  (=  19-2784  mm.)  in  diameter. 

"  Length  of  Screw  of  Nose-piece  to  be  not  less  than  0.125  in.  (  = 
3.1750  mm.). 

"Limits 
"  Nose-piece: 

"  Core  Diameter  of  Thread  (A)  not  to  exceed  0.7674  in.  (=  19.4918 
mm.),  or  be  less  than  0.7644  in.  (=  19.4156  mm.). 

"  Full  Diameter  of  Thread  (B)  not  to  exceed  0.803  in.  (=  20.3960 
mm.),  or  be  less  than  0.800  in.  (=  20.3198  mm.). 
"  Objective:  — 

"Full  Diameter  of  Thread  (C)  at  top  of  thread  not  to  exceed  0.7982 
in.  (=  20.2741  mm.),  or  to  be  less  than  0.7952  in.  (20.1979  mm.). 


io6  ROYAL  MICROSCOPICAL  SOCIETY  STANDARDS       [Cn.  Ill 

"  Core  Diameter  of  Thread  (D)  at  bottom  of  thread  not  to  exceed 
0.7626  in.  (=19.3699  mm.),  or  to  be  less  than  0.7596  in.  (  =  19.2937 
mm.)." 

§  176.  Royal  Microscopical  Society  standards  for  eye-pieces  and 
substage. — The  standards  adopted  in  1899  were  four  in  number, 
but  in  actual  practice  only  two  are  used: 

Small  or  Continental  size,  0.917  in.  =  23.300  mm. 

Large  size,  1.27    in.  =  32.258  mm. 

The  size  here  given  is  the  internal  diameter  of  the  draw-tube,  the 
tightness  of  the  fit  being  left  to  the  manufacturer. 

Standard  size  for  substage  fitting,  1.527  in.  =  38.786  mm. 

The  gauges  for  the  above  sizes  have  been  deposited  at  the  National 
Physical  Laboratory,  and  maker's  gauges  may  be  compared  with 
the  standards  on  payment  of  a  small  fee. 

For  Collateral  Reading,  see  the  list  at  the  end  of  Chs.  I  and  II. 
and: 

\ELSON.  E.  M.  —  On  the  Origin  of  the  Society  Screw.    Jour.  Roy.  Micr.  Soc.,  1910 

P-  443- 

BROCHER,  F.  —  Jour.  Roy.  Micr.  Soc.,  1912,  p.  244.  In  this  discussion  it  is  shown 
that  the  weariness  in  using  the  microscope  is  due  to  the  effort  to  make  the 
image  sharp  by  eye  accommodation  instead  of  by  the  fine  adjustment. 


CHAPTER  IV 

INTERPRETATION  OF  APPEARANCES 

§  186.     Apparatus  and  material  for  Chapter  IV. 

1.  Laboratory    compound     micro-  8.  Solid  glass  rod  and  glass  tube 
scope  (fig.  25).  (§  202). 

2.  Preparation  of  fly's  wing  (§  188).  9.  Collodion  (§204). 

3-  5o%  glycerin  (§  202).  10.  Carmine,    starch,    chalk    dust 

4.  Slides  and  covers  (Ch.  X).  (§  206). 

5.  Preparation  of  letters  in  stairs  n.  Frog  (§  209). 

(fig.  66).  12.  Castor  oil  (§  209). 

6.  Gum    arabic    mucilage    for    air  13.  Micro-polariscope  (Ch.  VIII). 
bubbles  (§  194).  14.  Fine  forceps  (fig.  70). 

7.  Oil    or    milk    for    oil    globules  15.  Eosin  (§  202). 
(§ 


§  187.  Appearances  which  seem  perfectly  unmistakable  with  a 
low  power  may  be  found  erroneous  or  very  inadequate  with  high 
powers;  for  details  of  structure  which  cannot  be  seen  with  a  low  power 
may  become  perfectly  evident  with  a  higher  power  or  a  more  perfect 
objective.  On  the  other  hand,  the  problems  of  microscopic  structure 
become  more  and  more  complex  with  increased  precision  of  investi- 
gation and  more  perfect  optical  appliances,  for  structures  that  appeared 
intelligible  with  a  less  perfect  microscope  may  show  complexities  in 
their  details  of  structure  with  the  more  perfect  microscope  which 
open  up  an  entirely  new  field  for  interpretation. 

One  must  always  be  on  the  lookout  for  errors  in  judgment  induced 
by  color  effects  due  to  purely  optical  means  and  to  color  in  the  speci- 
men and  also  to  avoid  confusing  refraction,  reflection,  and  diffraction 
effects  with  pigments,  or  actual  structures  of  any  kind.  It  is  not  infre- 
quent in  searching  for  malarial  pigment  in  the  red  blood  corpuscles 
to  mistake  the  dark-looking  crenations  on  the  corpuscles  for  the 
pigment  sought  (§  iSya). 

The  need  of  the  most  careful  observation  and  constant  watchful- 
ness lest  the  appearances  may  be  deceptive  is  thus  admirably  stated 

107 


108         INTERPRETATION  OF  APPEARANCES       [Cn.  IV 

by  Dallinger  (see  Carpenter-Dallinger,  p.  427):  "The  correctness  of 
the  conclusions  which  the  microscopist  will  draw  regarding  the  nature 
of  any  object  from  the  visual  appearances  which  it  presents  to  him 
when  examined  in  the  various  modes  now  specified  will  necessarily 
depend  in  a  great  degree  upon  his  previous  experience  in  microscopic 
observation  and  upon  his  knowledge  of  the  class  of  bodies  to  which  the 
particular  specimen  may  belong.  Not  only  are  observations  of  any 
kind  liable  to  certain  fallacies  arising  out  of  the  previous  notions 
which  the  observer  may  entertain  in  regard  to  the  constitution  of 
the  objects  or  the  nature  of  the  actions  to  which  his  attention  is  di- 
rected, but  even  the  most  practised  observer  is  apt  to  take  no  note 
of  such  phenomena  as  his  mind  is  not  prepared  to  appreciate.  Errors 
and  imperfections  of  this  kind  can  only  be  corrected,  it  is  obvious,  by 
general  advance  in  scientific  knowledge;  but  the  history  of  them  affords 
a  useful  warning  against  hasty  conclusions  drawn  from  a  too  cursory 
examination.  If  the  history  of  almost  any  scientific  investigation 
were  fully  made  known,  it  would  generally  appear  that  the  stability 
and  completeness  of  the  conclusions  finally  arrived  at  had  been  only 
attained  after  many  modifications,  or  even  entire  alterations,  of  doc- 
trine. And  it  is  therefore  of  such  great  importance  as  to  be  almost 
essential  to  the  correctness  of  our  conclusions  that  they  should  not  be 
finally  formed  and  announced  until  they  have  been  tested  in  every 
conceivable  mode.  It  is  due  to  science  that  it  should  be  burdened 
with  as  few  false  facts  [artifacts]  and  false  doctrines  as  possible.  It 
is  due  to  other  truth-seekers  that  they  should  not  be  misled,  to  the 
great  waste  of  their  time  and  pains,  by  our  errors.  And  it  is  due  to 
ourselves  that  we  should  not  commit  our  reputation  to  the  chance  of 
impairment  by  the  premature  formation  and  publication  of  conclu- 
sions which  may  be  at  once  reversed  by  other  observers  better 
informed  than  ourselves,  or  may  be  proved  fallacious  at  some  future 
time,  perhaps  even  by  our  own  more  extended  and  careful  re- 
searches. The  suspension  of  the  judgment  whenever  there  seems 
room  for  doubt  is  a  lesson  inculcated  by  all  those  philosophers  who 
have  gained  the  highest  repute  for  practical  wisdom;  and  it  is  one 
which  the  microscopist  cannot  too  soon  learn  or  too  constantly 
practise." 


CH.  IV]  INTERPRETATION  OF  APPEARANCES  109 

The  general  law  for  the  whole  matter  is  to  study  the  object  in  every 
way  possible  (§  218). 

For  the  experiments,  §  188-201,  no  condenser  is  to  be  used,  except 
in  a  part  of  §  201. 

§  187a.  "The  distinction  between  a  dark  element  which  is  referable  to 
pigment  and  a  dark  element  which  is  referable  to  the  deflection  of  light  can 
generally  be  made  by  watching  the  effect  produced  by  the  alteration  of  the 
focus.  Where  the  dark  element  corresponds  to  a  point  from  which  light  is 
deflected  a  change  of  the  focus  will  be  associated  with  a  change  from  dark  to 
bright.  Where  pigment  is  in  question  a  change  of  focus  will  substitute  only  a 
more  diffuse  for  a  less  diffuse  dark  element."  (Wright,  p.  44.) 

§  188.  Dust  or  Cloudiness  on  the  Ocular.  —  Employ  the  16  mm. 
objective,  4x  or  5x  ocular,  and  fly's  wing  as  object. 

Unscrew  the  field-lens  and  put  some  particles  of  lint  from  dark 
cloth  on  its  upper  surface.  Replace  the  field-lens  and  put  the  ocular 
in  position  (§45).  Light  the  field  well  and  focus  sharply.  The 
image  will  be  clear,  but  part  of  the  field  will  be  obscured  by  the  irregular 
outline  of  the  particles  of  lint.  Move  the  object  to  make  sure  this 
appearance  is  not  due  to  it. 

Grasp  the  ocular  by  the  milled  ring,  just  above  the  tube  of  the 
microscope  and  rotate  it.  The  irregular  objects  will  rotate  with  the 
ocular.  Cloudiness  or  particles  of  dust  on  any  part  of  the  ocular  may 
be  detected  in  this  way. 

Unscrew  the  field-lens  and  remove  the  lint  before  proceeding. 

§  139.  A  small  bright  field.  — With  low  objectives  (25-50  mm.), 
if  too  small  a  diaphragm  is  used  and  put  close  to  the  object,  only  the 
central  part  of  the  field  will  be  illuminated,  and  around  the  small 
light  circle  will  be  seen  a  dark  ring  (fig.  65).  If  the  diaphragm  is 
lowered  or  a  sufficiently  large  one  employed,  the  entire  field  will  be 
lighted  (see  also  §  90  for  diaphragms  with  the  condenser). 

§  190.     Relative  position  of  objects  or  parts  of  the  same  object.  - 
The  general  rule  is  that  objects  highest  up  come  into  focus  last  in 
focusing  up,  first  in  focusing  down. 

§  191.  Objects  having  plane  or  irregular  outlines.  —  As  object 
use  three  printed  letters  in  stairs  mounted  in  Canada  balsam  (fig.  66). 
The  first  letter  is  placed  directly  upon  the  slide,  and  covered  with  a 
small  piece  of  glass  about  as  thick  as  a  slide.  The  second  letter  is 


no 


INTERPRETATION  OF  APPEARANCES 


[CH.  IV 


placed  upon  this  and  covered  in  like  manner.     The  third  letter  is 
placed  upon  the  second  thick  cover  and  covered  with  an  ordinary 


FIG.  65. 


THE  MICROSCOPIC  FIELD  COMPLETELY  AND  ONLY  PARTLY 
ILLUMINATED. 


A     The  field  completely  illuminated;  a  net  micrometer  is  used  as  object. 
B     The  field  is  only  partly  illuminated;   the  same  net  micrometer  is  used  as 
object,  but  not  all  of  it  appears  in  the  partially  lighted  field. 

cover-glass.  The  letters  should  be  as  near  together  as  possible,  but 
not  overlapping.  Employ  the  same  ocular  and  objective  as  above 
(§  188). 

Lower  the  tube  till  the  objective  almost  touches  the  top  letter; 
then  look  into  the  microscope  and  slowly  focus  up.     The  lowest  letter 


FIG.  66.     LETTERS  IN  STAIRS  TO  DETERMINE  RELATIVE  POSITION  BY  FOCUS- 
ING UP  AND  DOWN. 

will  first  appear  and  then,  as  it  disappears,  the  middle  one  will  appear 
and  so  on.  Focus  down,  and  the  top  letter  will  first  appear,  then  the 
middle  one,  etc.  The  relative  position  of  objects  is  determined  exactly 
in  this  way  in  practical  work. 


CH.  IV]    DETERMINATION  OF  THE  CHARACTER  OF  OBJECTS    in 

For  example,  if  one  has  a  micrometer  ruled  on  a  cover-glass  0.15- 
0.25  mm.  thick,  it  is  not  easy  to  determine  with  the  naked  eye  which 
is  the  ruled  surface.  But  if  one  puts  the  micrometer  under  a  micro- 
scope and  uses  a  4  mm.  objective,  it  is  easily  determined.  The  cover 
should  be  laid  on  a  slide  and  focused  till  the  lines  are  sharp.  Now, 
without  changing  the  focus  in  the  least,  turn  the  cover  over.  If  it  is 
necessary  to  focus  up  to  get  the  lines  of  the  micrometer  sharp,  the  lines 
are  on  the  upper  side.  If  one  must  focus  down,  the  lines  are  on  the  un- 
der surface.  With  a  thin  cover  and  delicate  lines  this  method  of  deter- 
mining the  position  of  the  rulings  is  of  considerable  practical  importance. 

§  192.  Determination  of  the  form  of  objects.  —  The  procedure  is 
exactly  as  for  the  determination  of  the  form  of  large  objects.  That 
is,  one  must  examine  the  various  aspects.  For  example,  if  one  were 
placed  in  front  of  a  wall  of  some  kind,  one  could  not  tell  whether  it 
was  a  simple  wall  or  whether  it  was  one  side  of  a  building  unless  in 
some  way  one  could  see  more  than  the  face  of  the  wall.  In  other  words, 
in  order  to  get  a  correct  notion  of  any  body,  one  must  examine  more 
than  one  dimension,  —  two  for  plane  surfaces,  three  for  solids.  So 
for  microscopic  objects,  one  must  in  some  way  examine  more  than 
one  face.  To  do  this  with  small  bodies  in  a  liquid  the  bodies  may  be 
made  to  roll  over  by  pressing  on  one  edge  of  the  cover-glass.  And  in 
rolling  over  the  various  aspects  are  presented  to  the  observer.  With 
solid  bodies,  like  the  various  organs,  correct  notions  of  the  form  of 
the  elements  can  be  determined  by  studying  sections  cut  at  right 
angles  to  each  other.  The  methods  of  getting  the  elements  to  roll 
over,  and  of  sectioning  in  different  planes,  are  in  constant  use  in  His- 
tology, and  the  microscopist  who  neglects  to  see  all  sides  of  the  tissue 
elements  has  a  very  inadequate  and  often  a  very  erroneous  conception 
of  their  true  form. 

§  193.  Transparent  objects  having  curved  outlines.  —  The  success 
of  these  experiments  will  depend  entirely  upon  the  care  and  skill  used 
in  preparing  the  objects,  in  lighting,  and  in  focusing. 

Employ  a  4  mm.  or  higher  objective  and  an  8x  or  lox  ocular  for 
all  the  experiments.  It  may  be  necessary  to  shade  the  object  (§  140) 
to  get  satisfactory  results.  When  a  diaphragm  is  used  the  opening 
should  be  small  and  it  should  be  close  to  the  object. 


112    DETERMINATION  OF  THE  CHARACTER  OF  OBJECTS    [Cn.  IV 


§  194.  Air  bubbles.  —  Prepare  these  by  placing  a  drop  of  thin 
gum  arabic  mucilage  on  the  center  of  a  slide  and  beating  it  with  a 
scalpel  blade  until  the  mucilage  looks  milky  from  the  inclusion  of  air 
bubbles.  Put  on  a  cover-glass  but  do  not  press  it  down. 

§  196.  Air  bubbles  with  central  illumination.  —  Shade  the  object 
and  with  the  plane  mirror  light  the  field  with  central  light  (fig.  21  B). 

Search  the  prepara- 
tion until  an  air  bubble 
is  found  appearing 
about  i  mm.  in  diam- 
eter, get  it  into  the 
center  of  the  field,  and 
if  the  light  is  central 
the  air  bubble  will  ap- 
pear with  a  wide,  dark, 
circular  margin  and  a 
small  bright  center.  If 
the  bright  spot  is  not 
in  the  center,  adjust 
the  mirror  until  it  is. 

This  is  one  of  the 
simplest  and  surest 
methods  of  telling  when 
the  light  is  central  or 
axial  when  no  conden- 
ser is  used  (§  98). 

Focus  both  up  and 
down,  noting  that,  in 
focusing  up,  the  central 


FIG.  67.     OBLIQUE  ILLUMINATION  WITH  A 
MIRROR  AND  WITH  A  CONDENSER. 

A  The  light  is  shown  to  be  oblique  with  ray  c; 
rays  A  B  are  central.  The  arrows  indicate  the 
path  of  the  rays.  (For  the  objective  see  explan- 
ation of  fig.  35.) 

B  Abbe  condenser  with  an  eccentric  dia- 
phragm (D)  admitting  light  only  on  one  side. 

Axis     The  principal  optic  axis.    Ob  Objective. 

S.  Axis  Secondary  axis. 


spot  becomes  very  clear  and  the  black  ring  very  sharp.  On  elevating 
the  tube  of  the  microscope  still  more  the  center  becomes  dim,  and  the 
whole  bubble  loses  its  sharpness  of  outline. 

§  196.  Air  bubbles  with  oblique  illumination.  —  Remove  the 
substage  of  the  microscope  and  all  the  diaphragms.  Swing  the  mirror 
so  that  the  rays  may  be  sent  very  obliquely  upon  the  object  (fig.  67). 
The  bright  spot  will  appear  no  longer  in  the  center,  but  on  the  side 
away  from  the  mirror  (fig.  68  A). 


CH.  IVD    DETERMINATION  OF  THE  CHARACTER  OF  OBJECTS    113 

§  197.  Oil  globules.  —  Prepare  these  by  beating  a  small  drop  of 
clove  or  other  oil  with  gum  arabic  mucilage  on  a  slide  and  covering  as 
directed  for  air  bubbles  (§  194),  or  use  a  drop  of  milk  in  a  drop  of  water. 

§  198.  Oil  globules  with  central  illumination.  —  Use  the  same 
diaphragm  and  light  as  above  (§  195).  Find  an  oil  globule  appearing 
about  i  mm.  in  diameter.  If  the  light  is  central  a  bright  spot  will 
appear  in  the  center.  Focus  up  and  down  and  note  that  the  dark 
ring  is  narrower  than  with  air  and  that  the  bright 
center  of  the  oil  globule  is  clearest  last  in  focus- 
ing up. 

§  199.     Oil  globules  with  oblique  illumination. 

-  Remove  the  substage,  etc.,  as  above,  swing  the 

mirror  to  one  side  and  light,  with  oblique  light. 

The  bright  spot  will  be  eccentric,  and  will  appear 

to  be  on  the  same  side  as  the  mirror  (fig.  68). 

§  200.  Oil  and  air  together.  —  Make  a  pre- 
paration exactly  as  described  for  air  bubbles 
•  £  1 94 ) ,  and  add  at  one  edge  a  little  of  the  mixture 
of  oil  and  mucilage  (§  197);  cover  and  examine. 

The  substage  need  not  be  used  in  this  experi- 
ment.    Search  the  preparation  until  an  ah"  bubble 
and  an  oil  globule,  each  appearing  about  i  mm. 
in  diameter,  are  found  in  the  same  field  of  view. 
Light  first  with  central  light,  and  note  that,  hi 
focusing  up,  the  ah-  bubble  comes  into  focus  first 
and  that  the  central  spot  is  smaller  than  that  of 
the  oil  globule.     Then,  of  course,  the  black  ring  will  be  wider  hi  the 
air  bubble  than  in  the  oil  globule.     Make  the  light  oblique.    The 
bright  spot  in  the  air  bubble  will  move  away  from  the  mirror,  while 
that  in  the  oil  globule  will  move  toward  it  (fig.  68). 

As  the  air  bubble  is  of  less  refractive  index  than  the  mucilage  it 
will  act  like  a  concave  lens  (fig.  69),  while  the  oil  globule,  having  a 
greater  refractive  index  than  the  mucilage,  will  act  as  a  convex  lens 
(fig.  69,  §  2ooa). 

It  is  possible  to  distinguish  oil  and  air  optically,  as  described  above, 
only  when  quite  high  powers  are  used  and  very  small  bubbles  are 


FIG.  68.  SMALL 
Ant  BUBBLE  (A) 
AND  OIL  GLOBULE 
(O)  WITH  OBLIQUE 
LIGHT. 

The  arrow  indi- 
cates the  direction 
of  the  light. 


114    DETERMINATION  OF  THE   CHARACTER  OF  OBJECTS    [Cn.  IV 


selected  for  observation.  If  a  16  mm.  objective  is  used  instead  of  a 
4  mm.,  the  appearances  will  vary  considerably  from  that  given  above 
for  the  higher  power.  It  is  well  to  use  a  low  as  well  as  a  high  power. 
Marked  differences  will  also  be  seen  in  the  appearances  with  objectives 
of  small  and  of  large  aperture,  as  the  larger  aperture  takes  in  more 

oblique  rays  and  hence  the  black 
margin  is  narrowed  (§  202). 

§  200a.  It  should  be  remembered 
that  the  image  in  the  compound  micro- 
scope is  inverted  (fig.  20);  hence  the 
bright  spot  really  moves  toward  the 
mirror  for  air,  and  away  from  it  for  oil. 

§  201.     Air  and  oil  by  reflected 
light.  —  Use  the  same  preparation 
as  in  §  200.    Cover  the  diaphragm 
or  mirror  so  that  no  transmitted 
light  can  reach  the  preparation. 
The  oil  and  air  will  appear  like 
globules    of    silver    on    a    dark 
ground.    The  part  that  was  dark- 
est in  each  with  transmitted  light 
will  be  lighted,  and  the  bright  central  spot  will  be  somewhat  dark. 
Use  also  the  condenser  and  dark-ground  illumination  (§  123). 
Experiments  in  which  the  substage  condenser  is  used  (§  202-209). 
§  202.     Distinctness  of  outline.  —  In  refraction  images  this  depends 
on  the  difference  between  the  refractive  power  of  a  body  and  that 
of  the  medium  which  surrounds  it.     The  oil  and  air  were  very  distinct 
in  outline,  as  both  differ  greatly  in  refractive  power  from  the  medium 
which  surrounds  them,  the  oil  being  more  refractive  than  the  mucilage 
and  the  air  less  (fig.  69). 

Place  a  fragment  of  a  cover-glass  on  a  clean  slide,  and  cover  it  (fig. 
70).  Use  it  as  object  and  employ  the  16  mm.  objective  and  8x  or 
IQX  ocular.  The  fragment  will  be  outlined  by  a  dark  band.  Put 
a  drop  of  water  at  the  edge  of  the  cover-glass.  It  will  run  in  and 
immerse  the  fragment.  The  outline  will  remain  distinct,  but  the  dark 
band  will  be  somewhat  narrower.  Remove  the  cover-glass,  wipe  it 


FIG.  69.     AIR  BUBBLE  AND  OIL 
GLOBULE  IN  WATER. 

Axis     The  principal  optic  axis. 

F,  F  The  principal  foci  of  the  air 
and  oil.  As  the  air  is  less  refractive 
than  water  its  focus  is  virtual.  The 
focus  of  the  oil  globule  is  real,  as  its 
refraction  is  greater  than  water. 


CH.  IV]    DETERMINATION  OF  THE  CHARACTER  OF  OBJECTS    115 


FlG  70  FlNE 
FORCEPS  FOR  PLAC- 
™G 


dry,  and  wipe  the  fragment  and  slide  dry  also.  Put  a  drop  of  50% 
glycerin  on  the  middle  of  the  slide  and  mount  the  fragment  of  cover- 
glass  in  that.  The  dark  contour  will  be  much  narrower  than  before. 

Draw  a  solid  glass  rod  out  to  a  fine  thread. 
Mount  one  piece  in  air,  and  the  other  in  50% 
glycerin.  Put  a  cover-glass  on  each.  Employ 
the  same  optical  arrangement  as  before.  Ex- 
amine the  one  in  air  first.  There  will  be  seen  a 
narrow,  bright  band,  with  a  wide,  dark  band  on 
each  side  (fig.  71  a). 

The  one  in  glycerin  will  show  a  much  wider 
bright  central  band,  with  the  dark  borders  cor- 
respondingly narrow  (fig.  7ib).  The  dark  con- 
tour depends  also  on  the  numerical  aperture  of 
the  objective  —  being  wider  with  low  apertures. 
This  can  be  readily  understood  when  it  is  remem- 
bered that  the  greater  the  aperture  the  more  ob- 
lique the  rays  of  light  that  can  be  received,  and 
the  dark  band  simply  represents  an  area  in  which  the  rays  are  so 
greatly  bent  or  refracted  (fig.  69)  that  they  cannot  enter  the  objective 
and  contribute  to  the  formation  of  the  image;  the  edges  are  dark 

simply   because  no  light   from 
them  reaches  the  observer. 

If  the  glass  rod  or  any  other 
object  were  mounted  in  a  me- 
dium of  the  same  color  and  re- 
fractive power,  it  could  not  be 
distinguished  from  the  medium. 
The  effect  of  the  immersing 
liquid  on  the  contour  bands 
around  any  transparent  object 

is  made  of  practical  use  in  the  determination  of  the  refractive  index 
of  crystals.  When  the  crystal  and  liquid  are  of  the  same  index  there 
will  be  no  band,  and  the  more  they  differ,  the  wider  will  be  the  band. 
As  shown  in  §  194-201,  lighting  with  oblique  light,  also  focusing  up 
and  down,  will  indicate  whether  the  crystal  is  of  greater  or  less  index 


FIG.  71. 


GLASS  RODS  IN  AIR  AND 
IN  GLYCERIN. 


a  Glass  rod  in  air  and  viewed  by 
central  transmitted  light. 

b  Glass  rod  mounted  in  50%  gly- 
cerin ;  the  dark  border  is  narrower  than 
when  mounted  in  air. 


Il6    DETERMINATION  OF  THE   CHARACTER  OF  OBJECTS    [Cn.  IV 

than  the  liquid.  For  this  method  a  series  of  liquids  of  known  index 
of  refraction  must  be  at  hand.  For  a  complete  discussion,  see 
Chamot,  Ch.  IX. 

A  very  striking  and  satisfactory  demonstration  may  be  made  by 
painting  a  zone  or  band  of  eosin  or  other  transparent  color  on  a  solid 
glass  rod,  and  immersing  the  rod  in  a  test  tube  or  vial  of  cedar  oil, 

clove  oil,  or  turpentine.  Above 
the  liquid  the  glass  rod  is  very 
evident,  but  under  the  liquid  it 
can  hardly  be  seen  except  where 
the  red  band  is  painted  on  it. 
This  is  a  good  example  of  a  color 

FIG.  72.  SOLID  GLASS  ROD  COATED  image  and  of  a  refraction  image 
WITH  COLLODION  TO  SHOW  DOUBLE  ,  ,  /g 

CONTOUR.  to  the  naked  eye  (§ 


§  202a.  Some  of  the  rods  have  air  bubbles  in  them,  and  then  there  results 
a  capillary  tube  when  they  are  drawn  out.  It  is  well  to  draw  out  a  glass  tube 
into  a  fine  thread  and  examine  it  as  described.  The  central  cavity  makes  the 
experiment  much  more  complex. 

§  203.  Highly  refractive.  —  This  expression  is  often  used  in  de- 
scribing microscopic  objects  (medullated  nerve  fibers,  for  example), 
and  means  that  the  object  will  appear  to  be  bordered  by  a  wide,  dark 
margin  when  it  is  viewed  by  transmitted  light.  And  from  the  above 
(§  202),  it  would  be  known  that  the  refractive  power  of  the  object 
and  the  medium  in  which  it  was  mounted  must  differ  considerably. 

§  204.  Doubly  contoured.  —  This  means  that  the  object  is  bounded 
by  two  usually  parallel  dark  lines  with  a  lighter  band  between  them. 
In  other  words,  the  object  is  bordered  by  (i)  a  dark  line,  (2)  a  light 
band,  and  (3)  a  second  dark  line. 

This  may  be  demonstrated  by  coating  a  fine  glass  rod  (§  202)  with 
one  or  more  coats  of  collodion  or  celloidin  and  allowing  it  to  dry,  and 
then  mounting  in  50%  glycerin  as  above  (§  202).  Employ  a  4  mm. 
or  higher  objective,  light  with  transmitted  light,  and  it  will  be  seen 
that  where  the  glycerin  touches  the  collodion  coating  there  is  a  dark 
line,  next  this  is  a  light  band,  and  finally  there  is  a  second  dark  line 
where  the  collodion  is  in  contact  with  the  glass  rod  (fig.  72). 


CH.  IV]    DETERMINATION  OF  THE  CHARACTER  OF  OBJECTS    117 

§  204a.  The  collodion  used  is  a  6%  solution  of  soluble  cotton  in  equal 
parts  of  sulphuric  ether  and  95%,  or,  preferably,  absolute  alcohol.  It  is  well 
to  dip  the  rod  two  or  three  times  in  the  collodion  and  to  hold  it  vertically  while 
drying.  The  collodion  will  gather  in  drops,  and  one  will  see  the  difference 
between  a  thick  and  a  thin  membranous  covering  (fig.  72). 

§  205.  Optical  section.  —  This  is  the  appearance  obtained  in  ex- 
amining transparent  or  nearly  transparent  objects  with  a  microscope 
when  some  plane  below  the  upper  surface  of  the  object  is  in  focus. 
The  upper  part  of  the  object  which  is  out  of  focus  obscures  the  image 
but  slightly.  By  changing  the  position  of  the  objective  or  object, 
a  different  plane  will  be  in  focus  and  a  different  optical  section  obtained. 
The  most  satisfactory  optical  sections  are  obtained  with  high  objec- 
tives having  large  aperture. 

Nearly  all  the  transparent  objects  studied  may  be  viewed  in  optical 
section.  A  striking  example  will  be  found  in  studying  Mammalian 
red  blood  corpuscles  on  edge.  The  experiments  with  the  solid  glass 
rods  (fig.  71)  furnish  excellent  and  striking  examples  of  optical  sec- 
tions. 

§  206.  Currents  in  liquids.  —  Employ  a  16  mm.  objective,  and  as 
object  put  a  few  particles  of  carmine,  starch,  or  chalk  dust  on  the 
middle  of  a  slide  and  add  a  drop  of  water.  Grind  the  carmine  or 
other  substance  well  with  a  scalpel  blade;  leave  the  preparation 
uncovered.  If  the  microscope  is  inclined,  a  current  will  be  produced 
in  the  water,  and  the  particles  will  be  carried  along  by  it.  Note  that 
the  particles  seem  to  flow  up  instead  of  down;  why  is  this?  How 
would  it  appear  to  flow  with  an  erecting  microscope  (§  146,  i4Qa)? 

§  207.  Velocity  under  the  microscope.  —  In  studying  currents  or 
the  movement  of  living  things  under  the  microscope,  one  should  not 
forget  that  the  apparent  velocity  is  as  unlike  the  real  velocity  as  the 
apparent  size  is  unlike  the  real  size.  If  one  consults  fig.  29  it  will 
be  seen  that  the  actual  size  of  the  field  of  the  microscope  with  the  differ- 
ent objectives  and  oculars  is  inversely  as  the  magnification.  That 
is,  with  great  magnification  only  a  small  area  can  be  seen.  The  field 
appears  to  be  large,  however,  and  if  any  object  moves  across  the  field 
it  may  appear  to  move  with  great  rapidity,  whereas  if  one  measures 
the  actual  distance  passed  and  notes  the  time,  it  will  be  seen  that  the 
actual  motion  is  quite  slow.  One  should  keep  this  in  mind  in  studying 


il8  PEDESIS  OR  BROWNIAN  MOVEMENT  [Cn.  IV 

the  circulation  of  the  blood.  The  truth  of  what  has  just  been  said 
can  be  easily  demonstrated  in  studying  the  circulation  in  the  gills 
of  Necturus,  or  in  the  frog's  foot,  by  using  first  a  low  power  in  which 
the  field  is  actually  of  considerable  diameter  (fig.  29;  Table,  §  49) 
and  then  using  a  high  power.  With  the  high  power  the  apparent 
motion  will  appear  much  more  rapid.  For  spiral,  serpentine,  and  other 
forms  of  motion,  see  Carpenter-Dallinger,  p.  433. 

§  208.  Pedesis  or  Brownian  movement.  —  Employ  the  same 
object  as  above,  but  a  4  mm.  or  higher  objective  in  place  of  the  16 
mm.  Make  the  body  of  the  microscope  vertical  so  that  there  may  be 
no  currents  produced.  Use  a  small  diaphragm  and  light  the  field 
well.  Focus  and  there  will  be  seen  in  the  field  large  motionless  masses, 
and  between  them  small  masses  in  constant  motion.  This  is  an  in- 
definite, dancing,  or  oscillating  motion. 

This  indefinite  but  continuous  motion  of  small  particles  in  a 
liquid  is  called  Brownian  movement  or  Pedesis;  also,  but  improperly, 
molecular  movement,  from  the  smallness  of  the  particles. 

The  motion  is  increased  by  adding  a  little  gum  arabic  solution  or  a 
slight  amount  of  silicate  of  soda  or  soap;  sulphuric  acid  and  various 
saline  compounds  retard  or  check  the  motion.  One  of  the  best  ob- 
jects is  lamp-black  ground  up  in  water  with  a  little  gum  arabic.  Car- 
mine prepared  in  the  same  way,  or  simply  in  water,  is  excellent;  and 
very  finely  powdered  pumice-stone  in  water  has  for  many  years  been 
a  favorite  object.  Pedesis  is  exhibited  by  all  solid  matter  if  it  is  finely 
enough  divided. 

Compare  the  pedetic  motion  with  that  of  a  current  by  slightly 
inclining  the  tube  of  the  microscope.  The  small  particles  will  con- 
tinue their  independent  leaping  movements  while  they  are  carried 
along  by  the  current.  The  pedetic  motion  makes  it  difficult  to  obtain 
good  photographs  of  milk  globules  and  other  small  particles.  The 
difficulty  may  be  overcome  by  mixing  the  milk  with  a  very  weak 
solution  of  gelatin  and  allowing  it  to  cool  (10%  gelatin  is  good). 

Until  recently  no  adequate  explanation  of  this  movement  had  been 
offered.  At  the  present  time  it  is  believed  to  be  due  to  the  kinetic 
activity  of  matter,  and  in  itself  to  be  one  of  the  best  proofs  of  that 
activity.  This  is  what  is  said  by  Rutherford:  "The  character  of  the 


CH.  IV]  PEDESIS  OR  BROWNIAN  MOVEMENT  119 

Brownian  movement  irresistibly  impresses  the  observer  with  the  idea 
that  the  particles  are  hurled  hither  and  thither  by  the  action  of  forces 
resident  in  the  solution,  and  that  these  can  only  arise  from  the  con- 
tinuous and  ceaseless  movement  of  the  invisible  molecules  of  which 
the  fluid  is  composed."  "Whatever  may  be  the  exact  explanation 
of  this  phenomenon,  there  can  be  but  little  doubt  that  it  results  from 
the  movements  of  the  molecules  of  the  solution,  and  is  thus  a  striking, 
if  somewhat  indirect,  proof  of  the  general  correctness  of  the  kinetic 
theory  of  matter."  Nature,  Vol.  81,  1909,  pp.  257-263;  Science, 
N.  S.,  Vol.  30,  1909,  pp.  289-303. 

By  the  aid  of  the  ultra-microscope  it  has  been  shown  that  the 
particles  in  smoke,  etc.,  exhibit  the  pedetic  movement  even  more 
strikingly  than  do  those  in  liquids. 

§  209.  Demonstration  of  pedesis  with  the  polarizing  microscope 
(Ch.  VIII) .  —  The  following  demonstration  shows  conclusively  that 
the  pedetic  motion  is  real  and  not  illusive  (Ranvier,  p.  173). 

Open  the  abdomen  of  a  dead  frog  (an  alcoholic  or  formalin  speci- 
men is  satisfactory).  Turn  the  viscera  to  one  side  and  observe  the 
small  whitish  masses  at  the  emergence  of  the  spinal  nerves.  With 
fine  forceps  remove  one  of  these  and  place  it  on  the  middle  of  a  clean 
slide.  Add  a  drop  of  water,  or  of  water  containing  a  little  gum  arabic. 
Rub  the  white  mass  around  in  the  drop  of  liquid  and  soon  the  liquid 
will  have  a  milky  appearance.  Remove  the  white  mass,  place  a  cover- 
glass  on  the  milky  liquid,  and  seal  the  cover  by  painting  a  ring  of 
castor  oil  all  around  it,  half  the  ring  being  on  the  slide  and  half  on 
the  cover-glass.  This  is  to  avoid  the  production  of  currents  by 
evaporation. 

Put  the  preparation  under  the  microscope  and  examine  with  first 
a  low  power,  then  a  high  power  (4  mm.).  In  the  field  will  be  seen 
multitudes  of  crystals  of  carbonate  of  lime;  the  larger  crystals  are 
motionless,  but  the  smallest  ones  exhibit  marked  pedetic  movement. 

Use  the  micro-polariscope  (Ch.  VIII),  light  with  great  care,  and 
exclude  all  adventitious  light  from  the  microscope  by  shading  the 
object  (§  140)  and  also  by  shading  the  eye.  Focus  sharply  and  ob- 
serve the  pedetic  motion  of  the  small  particles,  then  cross  the  polarizer 
and  analyzer,  that  is,  turn  one  or  the  other  till  the  field  is  dark.  Part 


120  DARK-GROUND   ILLUMINATION  [Cn.  IV 

of  the  large  motionless  crystals  will  shine  continuously  and  a  part 
will  remain  dark,  but  small  crystals  between  the  large  ones  will  shine 
for  an  instant,  then  disappear,  only  to  appear  again  the  next  instant. 
This  demonstration  is  believed  to  furnish  absolute  proof  that  the 
pedetic  movement  is  real  and  not  illusory. 

For  tfye  help  given  by  the  micro-spectroscope  see  Ch.  VIII. 

§  210.  Use  of  dark-ground  illumination  for  interpreting  appearances. 
—  Dark-ground  illumination  is  almost  invaluable  for  bringing  out 
details  of  structure  and  for  showing  movement  in  living  things.  The 
granules  and  different  parts  in  living  cells  and  minute  organisms  are 
so  nearly  the  same  refractive  index  that  it  is  exceedingly  difficult  to 
differentiate  them  with  the  ordinary  methods  of  illumination.  On 
the  other  hand,  with  dark«-ground  illumination  the  different  structures 
stand  out  with  the  greatest  clearness. 

§  211.     Specimens  to  use  for  dark-ground  illumination.  — 

(i)  Organisms  from  hay  infusion.  Use  for  the  infusion  a  small 
fruit  jar  or  other  glass  dish.  Go  to  a  stream  or  pond  and  from  a  shal- 
low stagnant  pool  along  the  edge  take  some  of  the  surface  of  the  mud 
and  put  it  into  the  jar  with  some  of  the  water.  Add  some  of  the  dead 
grass  found  along  the  edge  of  the  pond,  cut  up  into  short  pieces.  Set 
in  a  warm  dimly  lighted  or  dark  place  for  a  day  or  longer.  This 
should  soon  be  alive  with  all  sorts  of  minute  living  things. 

If  it  is  not  easy  to  get  the  water,  mud,  and  dead  grass,  fairly  good  re- 
sults are  obtained  by  putting  some  ordinary  hay  in  water  of  any  kind. 

With  fine  forceps  take  a  leaf  or  piece  of  stem  of  the  dead  grass  and 
put  it  on  a  slide  i  mm.  thick.  Move  it  around  and  press  it  dowrn  so 
that  a  good  drop  of  liquid  and  debris  will  be  on  the  slide.  Remove 
the  grass  and  cover  the  liquid  with  a  0.15  mm.  cover-glass.  This 
should  be.studied  fresh  with  a  4  mm.  objective,  5x  or  lox  ocular,  and 
transmitted  light.  Now  put  in  place  the  dark-ground  illuminator, 
center  it  (§  126),  and  add  some  distilled  water  or  some  homogeneous 
liquid  to  the  top  of  the  condenser  and  run  it  up  till  the  liquid  is  in 
contact  with  the  under  side  of  the  slide. 

Put  a  drop  of  homogeneous  liquid  on  the  cover-glass  and  use  a 
homogeneous  immersion  objective  in  which  the  aperture  has  been 
cut  down  to  0.95  or  less.  Examine  as  directed  in  §  127-128. 


CH.  IV]  EXPERIMENTS  FOR  INTERPRETATION  121 

(2)  Saliva.     Put  a  drop  of  saliva  on  a  slide  i  mm.  thick  and  cover 
it  with  a  0.15  mm.  cover-glass.     Examine  as  in  (i). 

Note  the  pedetic  or  Brownian  movement  of  the  granules  in  the 
rounded  salivary  corpuscles,  the  minute  granules  in  the  broad  oval 
epithelium,  etc. 

(3)  Fresh  blood.     Make  a  preparation  of  fresh  human  blood  as 
follows:   Use  a  clean  slide  i  mm.  thick  and  have  ready  and  handy  a 
cover-glass  0.15  mm.  thick.     Wash  the  middle  ringer  well  with  soap 
and  water  and  wipe  it  dry  with  a  piece  of  gauze.     Then  wipe  it  again 
with  a  piece  of  gauze  wet  with  95%  alcohol. 

Sterilize  a  needle  by  heating  it  to  redness.  Make  two  or  three 
good  pricks  in  the  clean  finger  with  the  sterile  needle.  Squeeze  the 
finger  well  and  a  drop  of  blood  will  run  out.  Touch  this  blood  to  the 
middle  of  the  slide  and  cover  it  immediately.  Press  the  cover  down 
so  that  there  will  be  a  very  thin  layer  of  blood.  Examine  with  the 
dark-ground  illumination,  using  the  homogeneous  objective  with  re- 
duced aperture.  Use  homogeneous  liquid  to  join  the  slide  and  top  of 
the  condenser. 

The  appearance  of  a  fresh  blood  preparation  with  dark-ground 
illumination  will  be  a  revelation  to  one  who  has  studied  blood  only 
with  the  usual  transmitted  light.  The  white  corpuscles,  or  leucocytes 
are  very  striking  objects,  especially  the  polymorphonuclear  ones  with 
granules.  These  granules  show  the  pedetic  or  Brownian  movement 
well;  and  if  the  room  is  warm  where  the  work  is  done  the  amoeboid 
movement  is  very  striking.  For  the  blood  of  2  individuals  studied 
the  leucocytes  in  one  (male)  moved  6.8  /x.  per  minute;  in  the  other 
(female)  the  movement  was  18  ft  per  minute. 

In  addition  to  the  corpuscles  and  minute  granules  of  various  kinds 
one  is  almost  sure  to  see  the  fibrin  filaments  arranged  something  like 
a  spider's  web. 

§  212.  Difference  of  appearance  due  to  difference  of  focus.  — 
If  one  takes  a  geometrical  pattern  like  that  shown  in  fig.  73  and  looks 
at  it  in  the  ordinary  way  the  appearance  is  that  of  white  spots  on  a 
dark  field.  If  now  the  head  is  held  closer  and  closer  to  the  picture  an 
inversion  will  take  place  and  the  appearance  is  of  dark  spots  in  a  white 
field.  This  illustrates  how  difficult  it  is  to  determine  the  real  appear- 


122  EXPERIMENTS  FOR  INTERPRETATION  [CH.  IV 

ance  under  the  microscope  of  objects  having  geometrical  patterns 
and  especially  if  there  are  several  of  them  superimposed,  as  with  the 
wire  gauze  experiment  (§  216).  The  image  is  often  just  as  satis- 
factory in  one  focus  as  in  another,  although  the  appearance  changes 
very  markedly  in  the  two  positions. 

§  213.  Comparing  two  microscopic  fields  side  by  side.  —  It  is  so 
difficult  to  carry  in  the  mind  the  exact  appearance  of  any  structure 
or  complex  pattern/  that  many  efforts  have  been  made  to  have  the 

microscopic  images  side  by  side  so  that 
they  can  be  looked  at  at  the  same  time. 
This  has  been  accomplished  by  using 
two  microscopes  and  projecting  two  fields 
side  by  side,  as  can  be  done  by  having 
two  microscopes  like  the  one  shown  in 

%  113- 

Another  method  is  by  means  of  a  com- 
parison ocular  (fig.  74).     Then  two  objects 

FIG.  73.     GEOMETRICAL  under    two  microscopes  have    the   images 

PATTERN    TO    SHOW    DIF-  .                 . 

FERENCE  OF  APPEARANCE  side  by  side  in  the  ocular,  half  the  field 

DEPENDING  ON  THE  Focus,  being  taken  up  by  one  object  and  half  by 

(From  Sir  A.  R.  Wright's     tne  other;  then  the  eye  can  compare  two 
Microscopy). 

structures  side  by  side. 

§  214.  Muscae  volitantes.  —  These  specks  or  filaments  in  the 
eyes  due  to  minute  shreds  or  opacities  of  the  vitreous  humor  some- 
times appear  as  part  of  the  object  as  they  are  projected  into  the  field 
of  vision.  They  may  be  seen  by  looking  into  the  well-lighted  micro- 
scope when  there  is  no  object  under  the  microscope.  They  may 
also  be  seen  by  looking  at  brightly  illuminated  snow  or  other  white 
surface.  By  studying  them  carefully  it  will  be  seen  that  they  are 
somewhat  movable  and  float  across  the  field  of  vision,  and  thus  do 
not  remain  in  one  position  as  do  the  objects  under  observation.  Fur- 
thermore, one  may,  by  taking  a  little  pains,  familiarize  himself  with 
the  special  forms  in  his  own  eyes  so  that  the  more  conspicuous  at  least 
may  be  instantly  recognized. 

§  215.  Miscellaneous  observations.  —  In  addition  to  the  above 
experiments  it  is  very  strongly  recommended  that  the  student  follow 


CH.  IV] 


EXPERIMENTS  FOR  INTERPRETATION 


123 


the  advice  of  Beale,  p.  248,  and  examine  first  with  a  low  power  then 
with  a  higher  power;   mounted  dry,  then  in  water;   lighted  with  re- 


r 

LS  '. 

\       ~T 

b^"-—  -1 

7  1 

^--d 

fcnt 

IF      1 

TIG.  74.     COMPARISON  OCULAR  FOR  PLACING  HALF  THE  FIELDS  OF  Two 
MICROSCOPES  SIDE  BY  SIDE.    (Rl  R2). 

(Bausch  &  Lomb  Optical  Co.,  from  Chamot). 

Tl     To  fit  into  the  tube  of  the  left  microscope. 

T2     To  fit  into  the  tube  of  the  right  microscope. 

P2  Prism  to  reflect  the  beam  from  the  right  microscope  to  the  prism  R2, 
whence  it  is  reflected  up  through  the  ocular  (O)  into  the  right  half  of  the  field 
shown  above  in  the  face  view. 

PlRl  The  prism  and  left  half  of  the  field  shown  in  face  view  in  the  diagram 
at  the  top. 

fleeted  light,  then  with  transmitted  light,  the  following:  potato,  wheat, 
rice,  and  corn  starch  (easily  obtained  by  scraping  the  potato  and  the 
grains  mentioned);  bread  crumbs;  portions  of  feather  (portions  of 
feather  accidentally  present  in  histological  preparations  have  been 


124  EXPERIMENTS   FOR  INTERPRETATION  [CH.  IV 

mistaken  for  lymphatic  vessels  —  Beale,  288) ;  fibers  of  cotton,  linen, 
and  silk  (textile  fibers  accidentally  present  have  been  considered 
nerve  fibers,  etc.);  the  scales  of  butterflies  and  moths,  especially  the 
common  clothes  moths;  the  dust  swept  from  carpeted  and  wood  floors; 
tea  leaves  and  coffee  grounds ;  dust  found  in  living  rooms  and  places 
not  frequently  dusted  (in  the  last  will  be  found  a  regular  museum  of 
objects). 

§  216.  Wire  gauze  experiment.  —  For  a  very  striking  illustration 
of  the  need  of  care  in  interpretation  with  naked  eye  observation,  take 
two  pieces  of  wire  gauze  such  as  is  used  for  milk  strainers  or  some 
slightly  coarser.  Place  these  over  each  other  and  look  through  them 
toward  the  light.  Where  there  is  but  a  single  layer  the  weave  is 
evident,  but  where  the  two  pieces  overlap  the  appearance  is  very 
puzzling,  and  changes  constantly  as  one  piece  is  rotated,  bringing  the 
threads  and  meshes  at  an  angle.  One  could  hardly  believe  that  the 
structure  is  so  simple  when  looking  through  two  layers  of  the  gauze. 

If  it  is  necessary  then  to  see  all  sides  of  an  ordinary  gross  object, 
to  observe  it  in  various  positions  and  with  varying  illumination  and 
under  various  conditions  of  temperature,  moisture,  and  in  single  as 
well  as  multiple  layers  to  obtain  a  fairly  accurate  and  satisfactory 
knowledge  of  it,  so  much  the  more  is  it  necessary  to  be  satisfied  with 
the  interpretation  of  appearances  under  the  microscope  only  after 
applying  every  means  of  investigation  at  command.  Even  then 
only  such  details  of  the  image  will  be  noted  and  understood  as  the 
brain  behind  the  eye  has  been  trained  to  appreciate. 

§  216a.  Experiment  with  wire  gauze.  —  For  this  very  striking,  naked-eye 
experiment  with  the  wire  gauze  the  author  is  indebted  to  a  suggestion  from  Dr. 
Chamot. 

§  217.  Inversion  of  the  microscopic  image.  —  As  all  the  images 
produced  by  the  modern  compound  microscope  are  inverted  unless 
they  are  erected  by  a  special  arrangement  of  prisms,  one  must  learn 
to  interpret  the  appearances  in  an  inverted  image  with  the  same 
certainty  as  in  erect  images  seen  by  the  naked  eye  or  through  the  simple 
microscope.  It  may  be  remarked  in  passing  that  with  the  compound 
microscope  the  image  is  actually  erect  on  the  retina  of  the  eye  (fig. 
3,  20). 


CH.  IV]  EXPERIMENTS  FOR  INTERPRETATION  125 

With  the  compound  microscope  it  soon  becomes  as  easy  to  move 
the  slide  in  the  right  direction  to  see  a  desired  part  as  it  is  to  make  the 
proper  motions  when  examining  an  object  with  the  naked  eye,  al- 
though the  motions  are  directly  opposite  in  the  two  cases.  Indeed 
so  natural  does  it  become  for  the  worker  with  the  compound  micro- 
scope to  make  the  proper  motions  for  the  object  giving  the  inverted 
image,  that  if  he  uses  a  compound  microscope  with  an  erecting  prism 
he  almost  invariably  moves  the  preparation  in  the  wrong  direction 
(§  i49a).  With  the  simple  microscope,  however,  it  seems  like  naked- 
eye  observation  and  there  is  never  any  difficulty. 

This  goes  to  show  that  by  experience  it  is  as  easy  to  interpret 
inverted  as  erect  images.  This  is  further  illustrated  by  the  printer 
who  learns  to  read  type  without  difficulty,  although  it  is  a  great  puzzle 
to  one  who  has  only  learned  to  read  the  appearances  after  the  type 
has  been  printed  on  paper. 

§  218.  Summary  for  proper  interpretation.  —  To  summarize  this 
chapter  and  leave  with  the  beginning  student  the  result  of  the  experi- 
ence of  many  eminent  workers: 

(1)  Get  all  the  information  possible  with  the  unaided  eye.     See 
the  whole  object  and  all  sides  of  it,  so  far  as  possible. 

(2)  Examine  the  preparation  with  a  simple  microscope  in  the 
same  thorough  way  for  additional  detail. 

(3)  Use  a  low  power  of  the  compound  microscope. 

(4)  Use  a  higher  power. 

(5)  Make  sure  that  the  mirror  is  in  the  best  position  to  give  the  most 
favorable  light.     Vary  the  aperture  by  opening  and  closing  the  iris  dia- 
phragm to  find  the  aperture  which  gives  the  clearest  image  in  each  case. 

(6)  Shade  the  top  of  the  stage  of  the  microscope  to  cut  off  the 
light  from  above  and  thus  avoid  confusion  from  that  source. 

(7)  Use  the  highest  power  available  and  applicable.     In  this  way 
one  sees  the  object  as  a  whole  and  progressively  more  and  more  details.  * 

(8)  If  one  has  the  apparatus  it  is  a  good  plan  to  examine  specimens 
with  a  binocular  microscope  to  gain  the  best  notion  possible  of  the 
relative  position  of  parts  of  the  specimen. 

(9)  Use  the  dark-ground  illuminator  (§  210),  the  spectroscope,  and 
polariscope  (Ch.  VIII). 


126  EXPERIMENTS  FOR  INTERPRETATION  [Cn.  IV 

(10)  Try  staining  the  preparations  to  be  studied  in  various  ways 
to  bring  out  the  structural  details;  remember  also  the  advantage  of 
a  color  picture  over  a  pure  refraction  image  (§  137)  and  especially  of 
a  combined  color  and  refraction  image.  Keep  in  mind  also  that  the 
microscopic  image  cannot  be  expected  to  reveal  structural  details 
that  are  not  in  some  way  clearly  differentiated  in  the  specimen. 

(n)  If  artificial  light  must  be  used,  employ  a  screen  of  daylight 
glass  (§  92)  between  the  source  of  illumination  and  the  microscope; 
then  one  can  obtain  true  color  effects. 

(12)  The  composite  picture  derived  from  all  available  means  of 
observation  is  much  more  likely  to  be  correct  than  that  obtained  by 
only  one  or  two  means  of  observation. 

(13)  According  to  Wright,  p.  46,  it  is  far  more  difficult  to  prepare 
and  properly  illuminate  a  specimen  than  to  get  a  good  image  of  it 
after  it  is  thus  prepared  and  lighted. 

COLLATERAL  READING  FOR  CHAPTER  IV 

For  general  discussions:  Carpenter-Dallinger;  A.  E.  Wright,  Principles  of 
Microscopy,  Ch.  V;  Beale;  Spitta,  Microscope,  Ch.  XVIII;  Chamot,  Chemi- 
cal Microscopy. 

For  pedesis  see  Jevons  in  Quart.  Jour.  Science,  n.s.,  Vol.  VIII  (1878),  p.  167; 
Rutherford,  Science,  N.  S.  Vol.  XXX,  1909,  pp.  289-302.  For  the  original 
account  of  this  see  Robert  Brown,  "Botanical  appendix  to  Captain  King's 
voyage  to  Australia,"  Vol.  II,  p.  534  (1826). 

For  overcoming  pedesis  for  photography  see  Gage,  The  use  of  a  solution  of 
gelatin  to  obviate  pedesis  in  photographing  milk  globules  and  other  minute 
objects  in  water,  Transactions  Amer.  Micr.  Soc.,  Vol.  XXIV,  1902,  p.  21. 

For  figures  (photo-micrographs,  etc.)  of  the  various  forms  of  starch,  see 
Bulletin  No.  13  of  the  Chemical  Division  of  the  U.  S.  Department  of  Agri- 
culture. For  hair  and  wool,  see  Bulletin  of  the  National  Association  of  Wool 
Growers,  1875,  P-  47°;  Proc.  Amer.  Micro.  Soc.,  1884,  pp.  65-68;  Herzfeld, 
translated  by  Salter,  The  technical  testing  of  yarns  and  textile  fabrics,  London, 
1898. 

For  different  appearances  due  to  the  illuminator,  see  Nelson,  in  Jour.  Roy. 
Micr.  Soc.,  1891,  pp.  90-105;  and  for  the  illusory  appearances  due  to  diffrac- 
tion phenomena,  see  Carpenter-Dallinger,  p.  434;  Mercer,  Trans.  Amer. 
Micr.  Soc.,  V.  18  p.  321-396;  also,  A.  E.  Wright's  Principles  of  Microscopy, 
especially  the  first  five  chapters;  and  chapter  IX  and  the  appendix.  Conrad 
Beck.  The  Theory  of  the  Microscope.  Cantor  Lectures  before  the  Royal 
Society  of  Arts,  Nov.  Dec.,  1907.  59  pages,  London,  1908. 


CHAPTER  V 
MAGNIFICATION   AND    MICROMETRY 

§  225.     Apparatus  and  material  for  Chapter  V. 

1.  Simple    and    compound    micro-  5.  Stage    micrometer    (§  233). 
scope    (§228).  6.  Wollaston  camera  lucida  (§  234). 

2.  Block    for    magnifier    and    com-  7.  Ocular  screw   micrometers  and 
pound  microscope  (§  230,  236).  fixed  ocular  micrometers  (§  238,  241). 

3.  Steel  scale  or  rule  divided  into  8.  Necturus   red    blood    corpuscles 
millimeters  and  I  mm.   (§  231).  (§  248). 

4.  Dividers    (§  231).  9.  Eikonometer  (§  253). 

WHY  A  MAGNIFIED  IMAGE  is  NECESSARY 

§  226.  The  fundamental  reason  for  using  a  microscope  lies  in  the 
structure  of  the  eye  and  its  possibilities  of  adjustment  for  objects 
at  different  distances. 

The  sensory  receptors  or  neuro-epithelium  (rods  and  cones)  of  the 
eye  stand  in  general  with  their  long  axes  parallel  with  the  rays  of  light 
entering  the  eye,  hence  the  image  of  any  external  object  falls  ^on  the 
ends  of  the  sensory  receptors.  Now  it  is  believed  that  if  any  image 
falls  wholly  upon  one  of  the  receptors  it  will  appear  as  a  point,  and  if 
the  image  of  two  objects  close  together  were  to  fall  on  one  receptor 
the  two  objects  would  appear  as  one. 

§  227.  Robert  Hooke  (1674),  in  dealing  with  the  power  of  the  hu- 
man eye  to  distinguish  double  stars  and  to  see  two  points  or  two 
details  of  an  object  as  two,  concluded  that  the  two  stars  or  the  two 
points  of  any  object  must  at  be  least  far  enough  apart  to  make  the 
visual  angle  one  minute.  A  few  people  can  distinguish  double  stars 
with  a  visual  angle  less  than  one  minute,  but  for  many  people  the 
visual  angle  must  be  greater.  If  the  visual  angle  is  too  small,  then 
the  two  stars  or  two  points  appear  to  fuse  and  form  one.  The  visual 
angle  of  one  minute  then  does  not  represent  the  limit  of  visibility,  but 
the  limit  of  resolution,  that  is,  seeing  two  objects  as  two  separate  things, 

127 


128 


THE  MICROSCOPE  AND  VISUAL  ANGLE 


[CH.  V 


Now  as  the  visual  angle  under  which  any  given  object  is  seen 
depends  upon  its  distance  from  the  eye,  and  the  power  of  accommo- 
dation for  distance  in  the  eye  is  limited,  if  very  small  objects 
are  to  be  seen,  or  the  parts  of  larger  objects  are  to  be  distinguished  as 


FIG.  75.     CONSTANT  RETINAL  IMAGE  (R  /)  AND  CONSTANT  VISUAL  ANGLE 
WITH  VARYING  SIZE  OF  OBJECT  AT  DIFFERENT  DISTANCES. 

R  I  Retinal  image.  To  keep  this  of  constant  size  the  visual  angle  must 
remain  constant. 

Object  The  object  varying  in  size  directly  as  the  radius  to  keep  the  visual 
angle  and  the  retinal  image  constant. 

The  radii  in  this  figure  are  in  the  proportion  of  i,  2,  4. 

separate  details,  there  must  be  some  means  of  enabling  the  eye  to  get 
very  close  to  the  object. 

The  microscope  serves  to  increase  the  visual  angle  under  which  an 
object  is  seen,  thus  virtually  making  it  possible  to  get  the  eye  very 
close  to  the  object  and  still  retain  the  sharpness  of  the  retinal  image. 


CH.  V] 


THE  MICROSCOPE  AND  VISUAL  ANGLE 


129 


Or  to  put  it  in  another  way,  the  microscope  helps  the  eye  to  produce 
a  larger  retinal  image,  and  makes  the  details  large  enough  to  fall  on 
more  than  one  of  the  retinal  elements,  thus  making  resolution  possible. 
The  sensory  receptors  of  the  retina  —  the  rods  and  cones  —  are  quite 
close  together  and  over  the  greater  part  of  the  retina  are  commingled, 
there  being  more  rods 
than  cones.  In  the  re- 
gion of  greatest  visual 
acuity  (fovea  centralis 
of  macula  lutea),  only 
cones  are  present.  In 
general  the  rods  are  2  ft 
and  the  cones  6  ft  in  di- 
ameter. In  the  fovea, 
however,  the  cones  are 
slender,  being  only 
about  2  ft  or  3  ft  in  di- 
ameter. These  sizes 
give  a  clue  to  the  size 
the  retinal  image  must 
have  in  order  that  there 


FIG.  76.  CONSTANT  SIZE  OF  OBJECT,  THE  VIS- 
UAL ANGLE  AND  THE  RETINAL  IMAGE  VARYING 
WITH  THE  DISTANCE. 


R  I  The  retinal  image  varying  inversely  as 
the  distance  of  the  object. 

V  A  The  visual  angle  varying  with  the  dis- 
tance of  the  object  from  the  eye. 

Object  The  object  of  constant  size  but  varying 
distance  from  the  eye.  The  distance  of  the  object 
is  in  the  ratio  of  i,  2,  4.  The  entire  circle  is 
shown  at  the  right,  but  only  a  small  arc  in  the 
other  figures. 


be  resolution,  that  is, 
that  two  points  appear 
as  two  or  two  lines  ap- 
pear as  two. 

If  we  assume  that 
Hooke  was  correct  in 
the  assumption  that  for 
two  points  to  appear  as  two  a  visual  angle  of  i  minute  is  necessary, 
the  diameter  in  millimeters  or  inches  of  the  object,  or  the  separation 
of  the  two  points  to  render  them  visible  as  two,  is  easily  determined 
as  follows: 

The  nodal  point  or  optic  center  of  the  eye  is  considered  to  be  at 
the  center  of  a  circle  (fig.  75),  and  the  object  at  the  circumference. 
No  matter  how  great  or  how  small  the  visual  distance,  the  object  must 
subtend  one  minute  of  the  arc  of  the  circle  at  whose  circumference  it 


130  THE  MICROSCOPE  AND   VISUAL  ANGLE  [Cn.  V 

is  situated,  in  order  that  its  two  extremities  shall  appear  separate. 
And  so  with  any  two  details;  they  must  be  far  enough  apart  to  make 
the  visual  angle  one  minute. 

To  determine  the  actual  length  in  millimeters  required  to  subtend 
one  minute  of  arc  in  any  case,  it  is  only  necessary  to  remember  that 
the  entire  circumference  is  6.2832  times  its  radius  (2  TTT),  and  that 
this  circumference  is  divided  into  360°  or  21,600  minutes. 

If,  now,  the  radius  of  the  circle,  or  the  distance  of  the  eye  from 
the  object,  is  i  meter,  the  circumference  of  the  circle  will  be  6.2832 
meters  or  6283.2  millimeters.  As  there  are  21,600  minutes  in  the 
entire  circumference,  the  actual  length  of  one  minute  with  a  circle 
having  a  radius  of  one  meter  is  6283.2  mm.  divided  by  21,600 
equals  0.29088  mm.  That  is,  the  eye  at  one  meter  distance  requires 
two  points  or  two  lines  to  be  separated  a  distance  of  0.29088  mm. 
in  order  that  they  may  be  seen  as  two  and  not  appear  to  be  fused 
together. 

It  is  assumed  by  workers  with  the  microscope  that  the  distance  of 
most  distinct  vision  for  adults  when  looking  at  objects  for  details  of 
structure  is  254  mm.  or  10  inches.  This  is  the  standard  distance 
selected  for  the  determination  of  magnifying  power  in  microscopy 
also. 

The  question  now  is,  how  large  a  retinal  image  will  be  formed  by 
an  object  giving  a  visual  angle  of  i  minute  at  the  standard  distance  of 
254  mm. 

First  must  be  found  the  actual  size  of  the  object  to  give  a  visual 
angle  of  i  minute  at  254  mm.  distance.  It  is  known  from  the  above 
calculation  that  for  one  meter  or  1000  mm.  the  object  must  have  a 
size  of  0.29088  mm.  Now  for  254  mm.  the  length  must  be  yV/ir  of 
this  number  or  0.07388352  mm.,  that  is,  a  little  more  than  one-fourth 
the  size  at  i  meter. 

Now  to  determine  the  size  of  the  retinal  image  at  254  mm.  image 
distance,  the  distance  from  the  center  or  nodal  point  of  the  eye  must 
be  known  as  well  as  the  image  distance  and  the  size  of  the  object.  The 
distance  of  the  retinal  image  from  the  nodal  point  is  assumed  to  be 
15  mm.  (Howell,  p.  306);  then  the  size  of  the  retinal  image  will  be: 
0.07388352:  x:  :  254:  15=  0.00436  mm.  or  4-36/x,  and  this  size  would 


CH.  V] 


MAGNIFICATION   BY  THE  MICROSCOPE 


13* 


make  the  image  fall  on  at  least  two  of  the  cones  of  the  fovea,  and 
therefore  there  would  be  resolution  and  any  two  points  would  appear 
as  two  and  not  as  one. 


MAGNIFICATION 

§  228.  The  magnification,  am- 
plification, or  magnifying  power  of 
a  simple  or  compound  microscope 
is  the  ratio  between  the  apparent 
and  real  size  of  the  object  examined. 
The  apparent  size  is  obtained  by 
measuring  the  virtual  image  (fig.  77- 
78).  For  determining  magnification 
the  object  must  be  of  known  length 
and  is  designated  a  micrometer 
(§  233).  In  practice  a  virtual  im- 
age is  measured  by  the  aid  of  some 
form  of  camera  lucida  (fig.  81,  100), 
or  by  double  vision  (§230).  As 
the  length  of  the  object  is  known, 
the  magnification  is  easily  deter- 
mined by  dividing  the  size  of  the 
image  by  the  size  of  the  object.  For 
example,  if  the  virtual  image  meas- 
ures 40  mm.  and  the  object  magni- 
fied, 2  mm.,  the  amplification  is 
40  -r-  2  =  20,  that  is,  the  apparent 
size  is  twentyfold  greater  than  the 
real  size. 

Magnification  is  expressed  in  di- 
ameters or  times  linear;  that  is,  but 
one  dimension  is  considered.  In 
giving  a  scale  at  which  a  micro- 
scopical or  histological  drawing  is 
made,  the  word  "magnification"  is 
of  multiplication:  thus,  X45O  upon 


FIG.  77.  SIMPLE  MICROSCOPE 
WITH  THE  VIRTUAL  IMAGE  AT  250 
MM.  FROM  THE  EYE. 

A  xis  The  principal  optic  axis  of 
the  microscope  and  of  the  eye. 

/  The  principal  focus  of  the  mi- 
croscope. 

A  B  The  object  just  above  the 
.  focus  (/). 

B2  A2  the  retinal  image;  it  is  in- 
verted. 

A3  B3  The  virtual  image  at  250 
mm.  from  the  eye;  it  is  erect. 

Cr     Cornea  of  the  eye. 

R  Single  refracting  surface  of  the 
schematic  eye. 

L    The  crystalline  lens  of  the  eye. 

frequently  indicated  by  the  sign 
a  drawing  means  that  the  figure 


132 


MAGNIFICATION  BY  THE  MICROSCOPE 


[CH.  V 


Axis     The  principal  optic  axis  of  the  microscope  and  of  the  eye. 

/,  /     Principal  focus  of  the  objective,  and  of  the  ocular,  r  im,  the  real  image 


formed  by  the  objective  just 

the  ocular. 

cr  The  cornea  of  the  eye. 
rs  The  single  refracting 
/  The  crystalline  lens  of 
r  i  The  retinal  image;  it 
The  tube  length  of  the  mi- 

limeters,   and   the  image  dis- 

250  millimeters.    For  more 

fig.  20. 


above  the  principal  focus  of 


surface  of  the  schematic  eye. 
the  eye. 
is  erect. 

croscope  (fig.  25)  is  160  mil- 
tance  of  the   virtual   image, 
complete    explanation    see 


Virtual 

»-»->-  — 


FIG.  78.     COMPOUND  MICROSCOPE  SHOWING  ALL  THE  IMAGES. 


CH.  V]  MAGNIFICATION  BY  THE  MICROSCOPE  133 

or  drawing  has  the  width  or  length  of  every  detail  450  times  as  great 
as  the  object. 

§  229.  Magnification  of  real  images.  —  In  this  case  the  magnifi- 
cation is  the  ratio  between  the  size  of  the  real  image  and  the  size  of 
the  object,  and  the  size  of  the  real  image  can  be  measured  directly. 
By  recalling  the  work  on  the  function  of  an  objective,  it  will  be  remem- 
bered that  it  forms  a  real  image  on  the  ground-glass  placed  on  the 
top  of  the  tube,  and  that  this  real  image  could  be  looked  at  with  the 
eye  or  measured  as  if  it  were  an  actual  object.  For  example,  suppose 
the  object  were  three  millimeters  long  and  its  image  on  the  ground- 
glass  measured  15  mm.,  then  the  magnification  is  15  +  3  =  5,  that  is, 
the  real  image  is  5  times  as  long  as  the  object.  The  real  images  seen 
in  photography  are  mostly  smaller  than  the  objects,  but  the  magnifi- 
cation is  designated  in  the  same  way  by  dividing  the  size  of  the  real 
image  measured  on  the  ground-glass  by  the  size  of  the  object.  For 
example,  if  the  object  is  400  millimeters  long  and  its  image  on  the 
ground-glass  is  25  millimeters  long,  the  ratio  is  25-7-  400  =  yV- 
That  is,  the  image  is  yV  as  long  as  the  object  and  is  not  magnified 
but  reduced.  In  marking  negatives,  as  with  drawings,  the  sign  of 
multiplication  is  put  before  the  ratio,  and  in  the  example  the  designa- 
tion is  XyV-  In  photography  (Ch.  VII)  and  when  using  the  magic 
lantern  and  the  projection  microscope  the  images  are  real,  and  may 
be  measured  on  the  screen  as  if  real  pictures  (fig.  79). 

§  230.  The  magnification  of  a  simple  microscope  is  the  ratio  be- 
tween the  virtual  image  (fig.  6,  77,  A3B3)  and  the  object  magnified 
(A^1).  To  obtain  the  size  of  this  virtual  image,  place  the  tripod 
magnifier  near  the  edge  of  a  support  or  block  of  such  a  height  that 
the  distance  from  the  upper  surface  of  the  magnifier  to  the  table  is 
250  millimeters. 

As  object,  place  a  scale  of  some  kind  ruled  in  millimeters  on  the 
support  under  the  magnifier.  Put  some  white  paper  on  the  table 
at  the  base  of  the  support  and  on  the  side  facing  the  light. 

Close  one  eye,  and  hold  the  head  so  that  the  other  will  be  near  the 
upper  surface  of  the  lens.  Focus  if  necessary  to  make  the  image  clear. 
Open  the  closed  eye  and  the  image  of  the  rule  will  appear  as  if  on  the 
paper  at  the  base  of  the  support.  Hold  the  head  very  still,  and  with 


MAGNIFICATION  BY  THE  MICROSCOPE 


[Cn.  V 


dividers  get  the  distance  betwee'n  any  two  lines  of  the  image.  This  is 
the  so-called  method  of  double  vision  in  which  the  microscope  image 
is  seen  with  one  eye  and  the  dividers  with  the  other,  the  two  images 
appearing  to  be  fused  in  a  single  visual  field. 

§  231.     Measuring  the  spread  of  the  dividers.  —  This  should  be 
done  on  a  steel  scale  divided  to  millimeters  and  J  mm. 


a   6  fdff 


Fig. 


FIG.  79.     REAL  IMAGE  FORMED  BY  A  PROJECTION  MICROSCOPE. 
(From  the  Essays  of  George  Adams). 

A  B  Mirror  reflecting  the  parallel  rays  of  the  sun  upon  the  condenser  (C  D). 

a  b  c  d  e  f     Parallel  beams  of  light. 

C  D  The  condenser. 

N  O  The  stage  of  the  projection  apparatus. 

E  F  The  object. 

G  H  The  projection  objective. 

L  M  The  screen  upon  which  the  real  image  is  shown. 

/  K  The  real  image  of  the  object  (E  F). 

As  J  mm.  cannot  be  seen  plainly  by  the  unaided  eye,  place  one 
arm  of  the  dividers  at  a  centimeter  line,  and  with  the  tripod  magnifier 
count  the  number  of  spaces  on  the  rule  included  between  the  points 
of  the  dividers.  The  magnifier  simply  makes  it  easy  to  count  the  space 
on  the  rule  included  between  the  points  of  the  dividers  —  it  does  not,, 
of  course,  increase  the  number  of  spaces  or  change  their  value. 

As  the  distance  between  the  points  of  the  dividers  gives  the  size  of 


CH.  V] 


MAGNIFICATION  BY  THE  MICROSCOPE 


135 


the  virtual  image  (fig.  77),  and  as  the  size  of  the  object  is  known,  the 
magnification  is  determined  by  dividing  the  size  of  the  image  by  the 
size  of  the  object.  Thus,  suppose  the  distance  between  the  two  lines 
at  the  limits  of  the  image  is  measured  by  the  dividers  and  found  on 
the  steel  scale  to  be  15  millimeters,  and  the  actual  size  of  the  space 
between  the  two  lines  of  the  object  is  2  millimeters,  then  the  mag- 
nification is  15-7-  2  =  7.5;  that  is,  the  image  is  7.5  tunes  as  long  or 
wide  as  the  object.  In  this  case  the  image  is  said  to  be  magnified  7.5 
diameters,  or  7.5  times  linear. 

The  magnification  of  any  simple  magnifier  may  be  determined 
experimentally  in  the  way  described  for  the  tripod  magnifier;  but  this 


Stage 
Micrometer 

O.lmm 
0.01mm 


FIG.  80.     STAGE  MICROMETER  RULED  ON  A  COVER-GLASS. 

The  tenths  millimeter  (o.i  mm.)  spaces  are   divided  by  short  lines  making 
the  whole  micrometer  one  with  o.i,  0.05,  and  o.oi  millimeters. 

method  is  of  course  only  possible  when  the  observer  has  two  good  eyes. 
If  he  has  but  one  eye,  or  his  eyes  are  very  unlike,  then  the  magnifica- 
tion can  be  determined  with  one  eye  by  using  a  camera  lucida  or  the 
eikonometer  (§  234,  253). 

§  232.  The  magnification  of  a  compound  microscope  is  the  ratio 
between  the  final  or  virtual  image  and  the  object  magnified. 

The  determination  of  the  magnification  of  a  compound  microscope 
may  be  made  as  with  a  simple  microscope  (§  230),  but  this  is  fatiguing 
and  unsatisfactory. 

§  233.  Stage,  or  object  micrometer.  —  For  determining  the  mag- 
nification of  a  compound  microscope  and  for  the  purposes  of  microm- 
etry,  it  is  necessary  to  have  a  finely  divided  scale  or  rule  on  glass  or  on 
metal.  Such  a  finely  divided  scale  is  called  a  micrometer,  and  for 
ordinary  work  one  mounted  on  a  glass  slide  (1X3  in.,  25  X  76  mm.) 
is  most  convenient. 


i36 


MAGNIFICATION    OF    THE    MICROSCOPE 


[Cn.  V 


The  spaces  between  the  lines  should  be  o.i  and  o.oi  mm.  (or  if  in 
inches,  o.oi  and  o.ooi  in.).     Micrometers  are  sometimes  ruled  on  the 

slide,  but  more  satisfactorily 
on  a  cover-glass  of  known 
thickness,  preferably  0.15- 
0.18  mm.  The  covers 
should  be  perfectly  clean 
before  ruling,  and  after- 
wards simply  dusted  off 
with  a  camel's  hair  duster, 
and  then  mounted,  lines 
downward  over  a  shellac  or 
other  good  cell  (see  Ch.X). 
If  one  rubs  the  lines  the 
edges  of  the  furrow  made 
by  the  diamond  are  liable  to 
be  rounded  and  the  sharp- 
ness of  the  micrometer  is 
lost.  If  the  lines  are  on  the 

slide  and  uncovered  one  cannot  use  the  micrometer  with  an  oil  im- 
mersion, as  the  oil  obliterates  the  lines.  Cleaning  the  slide  makes  the 
lines  less  sharp,  as  stated.  If  the  lines  are  coarse,  it  is  an  advantage 
to  fill  them  with  plumbago  or  graphite.  This  may  be  done  with 
some  very  fine  plumbago  on  the  end  of  a  soft  cork,  or  by  using  a  soft 
lead  pencil.  Lines  properly  filled  may  be  covered  with  balsam  and 
a  cover-glass  as  in  ordinary  balsam 
mounting  (Ch.  X). 

§  234.  Determination  of  magnifica- 
tion. — This  is  most  readily  accomplished 
by  the  use  of  some  form  of  camera  lucida, 
that  of  Wollaston  being  most  convenient, 
as  it  may  be  used  for  all  powers,  and  the 
determination  of  the  standard  distance 

of  250  millimeters  at  which  to  measure  the  images  is  readily  accom- 
plished (fig.  81). 

Employ  the  16  mm.  objective  and  a  4x  or  5x  ocular  with  a  stage 


FIG.  81.     WOLLASTON'S  CAMERA  LUCIDA. 


FIG.   82. 
ETER    WITH 
LINES. 


STAGE    MICROM- 
A  RING   ON  THE 


CH.  Vj  MAGNIFICATION    OF    THE    MICROSCOPE  137 

micrometer  as  object.  For  this  power  the  o.i  mm.  spaces  of  the 
micrometer  should  be  used  as  object.  Focus  sharply. 

It  is  somewhat  difficult  to  find  the  micrometer  lines.  To  avoid 
this  it  is  well  to  have  a  small  ring  enclosing  some  of  the  micrometer 
lines  (fig.  82).  The  light  must  also  be  carefully  regulated.  If  too 
much  light  is  used,  i.e.,  too  large  an  aperture,  the  lines  will  be  drowned 
in  the  light.  In  focusing  with  the  high  powers  be  very  careful.  Re- 
member the  micrometers  are  expensive  and  one  cannot  afford  to  break 
them.  As  suggested  above,  focus  on  the  edge  of  the  cement  ring  en- 
closing the  lines;  then,  in  focusing  down  to  find  the  lines,  move  the 
preparation  very  slightly,  back  and  forth.  This  will  bring  the  lines 
into  the  field  and  the  shadow  made  by  them  will  indicate  their  pres- 
ence, and  one  can  then  focus  until  they  are  sharp. 

After  the  lines  are  sharply  focused,  and  the  slide  clamped  in 
position,  make  the  tube  of  the  microscope  horizontal,  by  bending  the 
flexible  pillar,  being  careful  not  to  bring  any  strain  upon  the  fine 
adjustment  (fig.  25). 

Put  a  Wollaston  camera  lucida  (fig.  81)  in  position,  and  turn  the 
ocular  around  if  necessary  so  that  the  broad  flat  surface  may  face 
directly  upward,  as  shown  in  the  figure.  Elevate  the  microscope  by 
putting  a  block  under  the  base,  so  that  the  perpendicular  distance 
from  the  upper  surface  of  the  camera  lucida  to  the  table  is  250  mm. 
(§  236).  Place  same  white  paper  on  the  work-table  beneath  the 
camera  lucida. 

Close  one  eye,  and  hold  the  head  so  that  the  other  may  be  very 
close  to  the  camera  lucida.  Look  directly  down.  The  image  will 
appear  to  be  on  the  table.  It  may  be  necessary  to  readjust  the  focus 
after  the  camera  lucida  is  in  position.  If  there  is  difficulty  in  seeing 
both  dividers  and  image,  consult  Ch.  VI.  Measure  the  image  with 
dividers  and  obtain  the  power  exactly  as  above  (§  231). 

Thus:  suppose  two  of  the  o.i  mm.  spaces  were  taken  as  object 
and  the  image  is  measured  by  the  dividers,  and  the  spread  of  the 
dividers  is  found  on  the  steel  rule  to  be  9.4  millimeters,  the  mag- 
nification (which  is  the  ratio  between  size  "of  image  and  object)  is 
9.4  -r-  0.2  =  47.  That  is,  the  magnification  is  47  diameters,  or  47 
times  linear. 


138 


MAGNIFICATION    OF    THE    MICROSCOPE 


[CH.  V 


Put  the  8x  or  lox  ocular  in  place  of  the  4x  or  5x,  and  then  put  the 
camera  lucida  in  position.  Measure  the  size  of  the  image  with  di- 
viders and  a  rule  as  be- 
fore. The  power  will  be 
considerably  greater  than 
when  the  low  ocular  was 
used.  This  is  because  the 
virtual  image  (fig.  78)  seen 
with  the  high  ocular  is 
larger  than  the  one  seen 
with  the  low  one. 

Lengthen  the  tube  of 
the  microscope  50-60  mm. 
by  pulling  out  the  draw- 
tube.  Remove  the  camera 
lucida  and  focus;  then  re- 
place the  camera  and  ob- 
tain the  magnification.  It 
is  greater  than  with  the 
shorter  tube.  This  is  be- 
cause the  real  image  (fig. 
83)  is  formed  farther  from 
the  objective  when  the 
tube  is  lengthened,  and 
the  objective  must  be 
brought  nearer  the  object. 
The  law  is:  the  magnifica- 
tion varies  directly  with 
the  relative  distance  of  the 
image  and  object  from  the 
center  of  the  lens  (fig.  84) ; 
thus,  if  the  image  is  four 
times  as  far  from  the 
center  of  the  lens  as  the 


FIG.  83.     To  SHOW  THE  RELATIVE  POSITION 
OF  THE  OBJECT  AND  THE  REAL  IMAGE. 

The  farther  from  the  lens  the  object,  the 
nearer  to  it  will  be  the  real  image  (Object-a, 
Image-a;  and  Object-b,  Image-b). 

axis  The  principal  optic  axis  extended 
above  and  below. 

Secondary  axis  a  and  b  The  secondary  axes 
at  the  limits  of  the  respective  images,  and 
objects. 


object,  then  it  will  be  four  times  as  large  as  the  object,  and  if  it  is 
one-fourth  as  far  from  the  center  of  the  lens  as  the  object  it  will  be 
only  one-fourth  as  big  as  the  object,  and  so  on. 


CH.  V] 


MAGNIFICATION    OF    THE    MICROSCOPE 


139 


§  235.  Varying  the  magnification  of  a  micro- 
scope. —  There  are  five  ways  of  varying  the 
power  of  a  compound  microscope : 

(1)  By  using  a  higher  or  lower  objective. 

(2)  By  using  a  higher  or  lower  ocular. 

(3)  By  lengthening  or  shortening  the  tube 
of  the  microscope. 

(4)  By  increasing  or  diminishing  the  dis- 
tance at  which  the  virtual  image  is  projected 
(fig.  85). 

(5)  By  changing  the  relative  position  of  the 
combinations  in  an  adjustable  objective  (§31, 
134)  or  by  the  use  of  an  amplifier  (§  235a). 

§  235a.  Amplifier.  —  In  addition  to  the  methods  of 
varying  the  magnification  given  in  §  235,  the  magnifica- 
tion is  sometimes  increased  by  the  use  of  an  amplifier, 
that  is,  a  diverging  lens  or  combination  placed  between 
the  objective  and  ocular  and  serving  to  give  the  image- 
forming  rays  from  the  objective  an  increased  diverg- 
ence. An  effective  form  of  this  accessory  was  made 
by  Tolles,  who  made  it  as  a  small  achromatic  con- 
cavo-convex lens  to  be  screwed  into  the  lower  end  of 
the  draw-tube  (fig.  25)  and  thus  but  a  short  distance 
above  the  objective.  The  divergence  given  to  the 
rays  usually  increases  the  size  of  the  real  image  about 
twofold. 

§  236.  Standard  distance  at  which  the  vir- 
tual image  is  measured.  —  For  obtaining  the 
magnification  of  both  the  simple  and  the  com- 
pound microscope  the  directions  were  to  meas- 
ure the  virtual  image  at  a  distance  of  250 
millimeters.  This  is  because  some  standard 
distance  must  be  chosen  so  that  different 
workers  can  compare  their  results.  The  mag- 
nification could  be  found  at  almost  any  dis- 
tance, and  in  getting  the  magnification  of 
drawings  the  image  distance  is  rarely  exactly 
250  millimeters.  Whenever  the  magnification 
of  the  microscope  as  a  whole  or  of  the  objec- 
tive or  the  ocular  is  mentioned,  however,  it  is 


Image 


Object 

FIG.  84.     To    SHOW 

THAT    THE    SlZE   OF    THE 

REAL  IMAGE  DEPENDS 
UPON  ITS  RELATIVE 
DISTANCE  FROM  THE 
CENTER  OF  THE  OB- 
JECTIVE. 

Object  i  The  object 
one  unit  of  distance 
from  the  center  of  the 
lens  (CL). 

Image  i,  2, 3,  4  The 
image  four  units  of  dis- 
tance from  the  lens  and 
hence  four  times  as  long 
as  the  object. 


140 


MAGNIFICATION    OF    THE    MICROSCOPE 


[CH.  V 


always  understood  that  this  magnification  is  at  the  standard  distance 
of  250  mm.  The  necessity  for  the  adoption  of  some  common  stand- 
ard will  be  seen  at  a  glance  in  fig.  85,  where'  is  represented  graphi- 
cally the  fact  that  the  size  of  the  virtual  image  depends  directly  on 
the  distance  at  which  it  is  projected,  and  this  size  is  directly  propor- 
tional to  the  vertical  distance  from  the  apex  of  the  triangle,  of  which 

it  forms  a  base.  The 
distance  of  250  milli- 
meters has  been 
chosen  on  the  sup- 
position that  it  is  the 
distance  of  most  dis- 
tinct vision  for  normal 
adults  when  examin- 
ing details. 

In  preparing  draw- 
ings it  is  often  of  great 
convenience  to  make 
them  at  a  distance 
less  or  greater  than 
the  standard.  In  that 
case  the  magnification 
must  be  determined 
for  the  image  distance 
actually  used. 

237.  Magnification  and  relation  of  the  object  to  the  principal 
focus.  —  As  shown  by  figures  86  and  87,  independent  of  the  equivalent 
focus  of  the  simple  microscope  or  the  objective,  the  real  image  or  the 
virtual  image,  as  the  case  may  be,  will  be  larger  the  nearer  the  object 
is  to  the  principal  focal  point. 

In  figures  88  and  89  it  is  shown  also  that  if  the  object  or  the  real 
image  is  in  the  plane  of  the  principal  focus,  the  rays  emerging  from 
the  simple  microscope  or  the  ocular  will  be  in  parallel  bundles,  and 
when  projected  by  the  eye  must  also  be  in  parallel  bundles.  It  is 
further  shown  in  such  a  case  that  the  rays  emanating  from  any  point 
in  the  object  or  real  image  will  not  in  that  case  form  a  virtual  point 


FIG.  85.  DIAGRAM  TO  SHOW  THAT  THE  SIZE  or 
THE  VIRTUAL  IMAGE  DEPENDS  UPON  THE  PROJEC- 
TION DISTANCE. 

a     Size  of  image  at  a  projection  distance  of  25  cm. 

b     Image  at  35  cm. 

The  sizes  are  directly  as  the  projection  distances. 
C  The  camera  lucida  and  under  it  a  spectacle  lens 
to  aid  the  eye  in  focusing  the  pencil  point;  this  is 
only  needed  by  those  with  defective  eyes. 


CH.  V] 


MAGNIFICATION    OF    THE    MICROSCOPE 


141 


FIG.  86.  DIAGRAMS  TO  SHOW  THAT  THE  SIZE  OF  THE  REAL  IMAGE  OF  A  LENS 
DEPENDS  UPON  THE  DISTANCE  OF  THE  OBJECT  FROM  THE  PRINCIPAL  Focus. 

Axis  The  principal  optic  axis  extended  above  and  below.  A  B,  B  A  The 
object  and  the  inverted  real  image.  /,  /  The  principal  focus  above  and  below 
each  lens.  Lc  The  lens. 

The  object  is  the  same  size  in  the  two  cases,  but  the  images  differ,  depending 
upon  the  distance  of  the  object  from  the  principal  focus,  being  longer  the  nearer 
the  object  is  to  the  focus. 


FIG.  87.  DIAGRAM  TO  SHOW  THAT  THE  SIZE  OF  THE  VIRTUAL  IMAGE  OF  A  LENS 
DEPENDS  UPON  THE  DISTANCE  OF  THE  OBJECT  FROM  THE  PRINCIPAL  Focus. 

A  B,  A  B  The  object  and  the  virtual  image.  /  /  The  principal  focus. 
L  The  lens,  ep  The  eye-point,  c  The  single,  ideal  refracting  plane. 

As  with  real  images,  the  size  of  virtual  image  in  a  given  lens  depends  upon 
the  nearness  of  the  object  to  the  principal  focus. 


142 


MAGNIFICATION    OF    THE    MICROSCOPE 


[CH.  V 


focus  at  the  standard  distance  of  250  mm.,  as  shown  in  fig.  77,  but 
will  remain  parallel.  At  that  distance  then  the  image  on  the  retina 
would  be  a  diffusion  circle.  In  order  that  there  be  the  appearance 
of  a  point  focus  the  distance  must  be  great  enough  so  that  the  parallel 
rays  from  a  point  will  be  separated  less  than  one  minute  (§  226-227). 


FIG.  88-89.     DIAGRAMS  OF  SIMPLE  AND  COMPOUND  MICROSCOPES  WITH 
PARALLEL  BEAMS  EMERGING  ABOVE  AND  PROJECTED  BELOW. 

Axis     The  principal  optic  axis. 

Object     The  object. 

Objective     The  objective  of  the  compound  microscope. 

r  i     The  real  image  formed  by  the  objective. 

Ocular-Magnifier  The  ocular  and  magnifier  for  the  real  image  in  the  com- 
pound microscope,  and  for  the  object  in  the  simple  microscope. 

Eye-point     The  most  favorable  position  for  the  eye  of  the  observer. 

Below,  at  250  mm.,  the  usual  position  of  the  projected  image,  no  image  is 
formed  with  parallel  rays.  These  only  seem  to  come  from  a  point  at  a  dis- 
tance where  their  separation  is  less  than  one  minute  (§  226-227). 

Table  of  magnification  and  of  the  valuations  of  the  ocular  microm- 
eter. —  The  table  should  be  filled  out  by  each  student.  In  using 
it  for  Micrometry  and  Drawing  it  is  necessary  to  keep  clearly  in  mind 
the  exact  conditions  under  which  the  determinations  were  made,  and 
also  the  ways  in  which  variations  in  magnification  and  the  valuation 
of  the  ocular  miqrometer  may  be  produced. 


CH.  V] 


MEASURING    WITH    THE    MICROSCOPE 


143 


OCULAR                                 OCULAR 

4x  or  5x                                   8x  or  lox 

OBJECTIVE 

TUBE 

IN 

TUBE 

OUT 
—  MM. 

TUBE 

IN 

TUBE 

OUT 
MM. 

OCULAR  MICROMETER 
VALUATION 
TUBE  IN.     OUT  MM. 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

SIMPLE  MICROSCOPE.                 X 

OCULAR  MICROMETER  AND  ITS  VALUATION 

§  238.  This,  as  the  name  implies,  is  a  micrometer  to  be  used  in 
connection  with  an  ocular.  It  consists  of  rulings  on  a  cover-glass  of 
fixed  or  of  movable  lines. 

This  form  of  micrometer  is  placed  at  the  level  where  the  real  image 
is  formed,  i.e.,  at  the  level  of  the  ocular  diaphragm  of  all  oculars. 
With  positive  oculars  it  would  therefore  be  outside  the  ocular  (fig.  22) 
and  with  negative  or  Huygenian  oculars  between  the  lenses  (fig.  23- 
24).  The  image  of  the  object  under  the  microscope  appears  to  be 
directly  upon  or  immediately  under  the  ocular  micrometer,  and  hence 
the  number  of  spaces  on  the  ocular  micrometer  required  to  measure 
the  real  image  may  be  read  off  directly.  This,  however,  is  measuring 
the  size  of  the  real  image,  and  the  actual  size  of  the  object  can  only 


144  MEASURING    WITH    THE    MICROSCOPE  [Ce.  V 

be  determined  by  determining  the  ratio  between  the  size  of  the  real 
image  and  the  object.  In  other  words,  it  is  necessary  to  get  the  valua- 
tion of  the  ocular  micrometer  in  terms  of  a  stage  micrometer. 

§  239.  Valuation  of  the  ocular  micrometer.  —  This  is  the  value  of 
the  divisions  of  the  ocular  micrometer  for  the  purposes  of  micrometry, 
and  is  entirely  relative,  depending  on  the  magnification  of  the  real 
image  formed  by  the  objective;  consequently  it  changes  with  every 
change  in  the  magnification  of  the  real  image,  and  must  be  especially 
determined  for  every  change  modifying  the  real  image  of  the  micro- 
scope (§  235). 

It  will  be  seen  when  the  ocular  micrometer  valuation  is  found  for 
different  objectives,  that  the  greater  the  magnification  of  the  objective 
the  less  will  be  the  ocular  micrometer  valuation;  and  conversely, 
the  less  the  magnification  of  the  objective  the  greater  will  be  the  ocular 
micrometer  valuation. 

§  240.  Obtaining  the  ocular  micrometer  valuation  for  an  ocular 
micrometer  with  fixed  lines.  —  If  the  ocular  micrometer  is  on  a  cover- 
glass,  place  it  on  the  diaphragm  of  the  $x  or  lox  ocular  after  removing 
the  eye-lens.  Screw  the  eye-lens  back  in  place,  and  put  the  ocular 
in  the  tube  of  the  microscope.  Put  a  16  mm.  objective  in  place.  Use 
the  stage  micrometer  as  object.  Light  the  field  well  and  look  into 
the  microscope.  The  lines  of  the  ocular  micrometer  should  be  very 
sharply  defined.  If  they  are  not,  raise  or  lower  the  eye-lens  to  make 
them  so;  that  is,  focus, as  with  the  simple  magnifier. 

When  the  lines  of  the  ocular  micrometer  are  distinct,  focus  the 
microscope  (§  234)  for  the  stage  micrometer.  The  image  of  the 
stage  micrometer  appears  to  be  directly  under  or  upon  the  ocular 
micrometer. 

Make  the  lines  of  the  two  micrometers  parallel  by  rotating  the 
ocular  or  changing  the  position  of  the  stage  micrometer  or  both  if 
necessary,  and  then  make  any  two  lines  of  the  stage  micrometer  coin- 
cide with  any  two  on  the  ocular  micrometer  (fig.  90).  To  do  this  it 
may  be  necessary  to  pull  out  the  draw-tube  a  greater  or  less  distance. 
See  how  many  spaces  are  included  in  each  of  the  micrometers  (see 
fig.  90,  98). 

Divide  the  value  of  the  included  space  or  spaces  on  the  stage  microm- 


CH.  V] 


MEASURING    WITH    THE    MICROSCOPE 


145 


eter  by  the  number  of  divisions  on  the  ocular  micrometer  required  to 
include  them,  and  the  quotient  so  obtained  will  give  the  valuation  of 
the  ocular  micrometer.  For  example,  suppose  the  millimeter  is  taken 
as  the  unit  for  the  stage  micrometer  and  this  unit  is  divided  into  spaces 
of  o.i  and  o.oi  millimeters.  If  with  a  given  optical  combination  and 
tube-length  it  requires  10  spaces  on  the  ocular  micrometer  to  include 
the  real  image  of  o.i  millimeter  on  ^  B 

the  stage  micrometer,  obviously  one 
space  on  the  ocular  micrometer  in- 
cludes only  one-tenth  as  much,  or  o.  i 
mm.  -r-  10  =  o.oi  mm.  That  is,  each 
space  on  the  ocular  micrometer  in- 
cludes o.oi  of  a  millimeter  on  the 
stage  micrometer,  or  o.oi  millimeter 
of  the  length  of  any  object  under  the 
microscope,  the  conditions  remaining 
the  same.  Or,  in  other  words,  it  re- 
quires 100  spaces  on  the  ocular  mi- 
crometer to  include  i  millimeter  on 
the  stage  micrometer;  then,  as  be- 
fore, i  space  of  the  ocular  micrometer 
would  have  a  valuation  of  o.oi  milli- 
meter for  the  purposes  of  micrometry. 
The  size  of  any  minute  object  may  be 
determined  by  multiplying  this  valua- 
tion oij  one  space  by  the  number  of 
spaces  required  to  include  it.  For  example,  suppose  the  fly's  wing 
or  some  part  of  it  covered  8  spaces  on  the  ocular  micrometer;  it 
would  be  known  that  the  real  size  of  the  part  measured  o.oi  mm. 
X  8  =  0.08  mm.  or  80  \L  (§  246). 

Proceed  in  exactly  the  same  manner  to  get  the  ocular  micrometer 
valuation  when  using  any  objective  whether  it  is  of  higher  or  lower 
power  than  the  one  in  this  section. 

Any  Huygenian  ocular  may  be  used  as  a  micrometer  ocular  by 
placing  the  ocular  micrometer  at  the  level  of  the  ocular  diaphragm 
where  the  real  image  is  formed.  If  there  is  a  slit  in  the  side  of  the 


FIG.  90.  THE  IMAGES  OF  THE 
OCULAR  AND  OF  THE  STAGE 
MICROMETER,  SHOWING  HOW  TO 
ARRANGE  THE  LINES. 

o.m  Ocular,  s.m  Stage  mi- 
crometer lines. 

A  Lines  of  the  ocular  mi- 
crometer opposite  the  middle  of 
the  lines  of  the  stage  micrometer. 

B  Lines  of  the  ocular  mi- 
crometer at  the  right  side  of  the 
lines  of  the  stage  micrometer 
(compare  fig.  98). 


146 


MEASURING    WITH    THE    MICROSCOPE 


[CH.  V 


ocular  and  the  ocular  micrometer  is  mounted  properly  it  may  be 
introduced  through  the  opening  in  the  side.  This  was  a  common 
method  with  the  older  microscopes.  When  there  is  no  side  opening 
the  eye-lens  may  be  unscrewed  and  the  ocular  micrometer  on  a 
cover-glass  laid  upon  the  ocular  diaphragm. 

OCULAR  MICROMETER  WITH  MOVABLE  SCALE 
§  241.    This  form  is  a  Huygenian  ocular  with  a  five  millimeter 
scale    divided    into   twenty   one-fourth   millimeter   intervals.     The 

pitch  of  the  screw 
moving  the  scale  is  J 
mm.;  therefore  one 
complete  revolution  of 
the  drum  moves  the 
scale  one-fourth  of  a 
millimeter,  or  one  in- 
terval. The  drum  is. 
divided  into  100  equal 
divisions,  thus  ena- 
bling one  to  measure 
TTir  of  an  interval  on 
the  micrometer  scale. 
This  ocular  microm- 
eter combines  the  ad- 
vantages of  the  ocular 
micrometer  with  a 
fixed  scale  and  the 
filar  micrometer.  To  complete  the  measurement  of  an  object  not 
exactly  included  between  any  two  lines  of  the  scale,  the  drum  need 
be  revolved  only  partly  around. 

§  242.  Valuation  of  the  movable  scale  ocular  micrometer  (fig.  91). 
—  Use  a  4  mm.  objective  and  proceed  exactly  as  for  the  micrometer 
with  fixed  lines,  except  that  a  partial  stage  micrometer  space  can  be 
measured  by  rotating  the  drum  until  the  ocular  micrometer  exactly 
coincides  with  the  stage  micrometer.  Make  sure  that  the  lines  of  the 
two  micrometers  are  correctly  related,  as  shown  in  fig.  90  and  98.  One 


FIG.  91.     OCULAR  MICROMETER  WITH  MOVABLE 
SCALE  AND  RECORDING  DRUM. 

(From  the  Catalogue  of  the  Spencer  Lens  Co.). 

The  recording  drum  is  divided  into  100  equal 
divisions. 


CH.  V] 


MEASURING    WITH    THE    MICROSCOPE 


147 


can  then  count  up  the  number  of  spaces  on  the  ocular  micrometer 
required  to  measure  one  or  more  spaces  of  the  stage  micrometer. 
To  this  is  then  added  the  TJ-jj-  spaces  on  the  drum.  For  example 
suppose  that  three  o.oi  mm.  spaces  of  the  stage  micrometer  are  taken 
as  object,  and  that  it  requires 
seven  complete  spaces  of  the 
ocular  micrometer  and  T5A  on 
the  drum  to  include  the  three 
spaces  on  the  stage  micrometer; 
then  each  space  on  the  ocular 
micrometer  would  be  equal  to 
0.03  mm.  divided  by  7.50  =  0.004 
mm.  or  4  p,.  One  of  the  spaces 
on  the  drum  which  represents 
one-hundredth  of  an  interval  on 
the  ocular  micrometer  would 
have  a  valuation  under  these 
conditions  of  4  ju-  divided  by 
100  =  0.04  microns.  This  gives 
a  notion  of  the  minuteness  of 
the  object  which  can  be  meas- 
ured, and  of  the  smallness  of 
the  error  in  measuring  large  ob- 
jects, even  if  the  observation 
erred  in  getting  the  object  one 
or  more  of  the  drum  divisions 
too  large  or  too  small. 

For  an  actual  measurement 
with  this  ocular  micrometer,  see 
(i 


FIG.  92.  FIELD  OF  THE  MICROSCOPE 
SHOWING  THE  MOVABLE  SCALE  OF 
THE  HUYGENIAN  MICROMETER  OCU- 

LAR    (FlG.   Ql). 

The  arrow  indicates  that  the  scale  may 
be  moved  in  both  directions. 

0,5,10,15,20  These  figures  indicate 
the  20  spaces  in  groups  of  5.  Each 
space  represents  a  total  revolution  of 
the  screw  (screw  with  \  mm.  pitch). 
Each  of  the  100  divisions  on  the  drum 
(fig.  91)  represents  then  7-J-7  mm. 

Object  A  circular  object  covering  five 
of  the  micrometer  spaces,  and  the  drum 
shows  45  division  to  measure  the  partial 
space;  the  entire  object  then  measures 
5.45  divisions. 


One  would  proceed  exactly  as  above  for  getting  the  valuation  with 
any  other  objective. 

FILAR  OCULAR  MICROMETER 

§  243.     This  form  of  ocular  micrometer  usually  consists  of  a  Rams- 
den  ocular  with  fixed  cross  lines  and  a  movable  line  (fig.  94). 


148 


MEASURING    WITH    THE    MICROSCOPE 


[CH.  V 


For  obtaining  the  valuation  of  this  ocular  micrometer  proceed  as 
follows:  Employ  a  4  mm.  objective.  Carefully  focus  the  y^  mm. 
lines.  The  lines  of  the  ocular  micrometer  should  also  be  sharp; 
if  they  are  not  focus  them  by  moving  the  ocular  up  or  down  in  the 
sliding  tube.  Make  the  vertical  lines  of  the  ocular  micrometer  parallel 
with  the  lines  of  the  stage  micrometer  (fig.  90,  98).  Note  the  posi- 
tion of  the  graduated  drum  and  the  teeth  of  the  recording  comb,  and 
then  rotate  the  wheel  until  the  movable  line  traverses  one  space  on 

the  stage  microm- 
eter. Each  tooth 
of  the  recording 
comb  indicates  a 
total  revolution  of 
the  wheel,  and  by 
noting  the  number 
of  teeth  required 
and  the  gradua- 
tions on  the  wheel, 
the  revolutions  and 
part  of  a  revolu- 
tion required  to 
measure  the  o.oi 


FIG.  93.     FILAR  MICROMETER  OCULAR. 

(From  the  i6th  ed.  of  the  Catalogue  of  the  Bausch  & 
Lomb  Optical  Co.). 

This  is  a  Ramsden  ocular,  and  the  recording  drum  is 


divided  into  100  equal  divisions,  and  as  the  pitch  of  the  mm.  of  the  stage 
screw  is  0.5  mm.,  each  division  on  the  drum  represents  mirrnmpt*>r  ran 
an  actual  movement  of  0.005  mm.  of  the  movable  line. 

be     easily     noted. 

Measure  in  like  manner  4  or  5  spaces  and  get  the  average.  Suppose 
this  average  is  ij  revolutions  or  125  graduations  on  the  wheel,  to 
measure  the  o.oi  mm.  or  10  p,  (see  §  246),  then  one  of  the  graduations 
on  the  wheel  would  measure  xo/x  divided  by  125  =  o.o8/u,.  In  using 
this  valuation  for  actual  measurement,  the  tube  of  the  microscope 
and  the  objective  must  be  exactly  as  when  obtaining  the  valuation 
(see  §  235-242). 

The  valuation  of  the  filar  micrometer  can  be  obtained  for  any 
objective  by  proceeding  exactly  as  above  (see  §  252  for  measure- 
ment). 


CH.  V] 


MEASURING    WITH    THE    MICROSCOPE 


149 


MlCROMETRY 

§  244.     Micrometry  is  the  determination  of  the  size  of  objects  .by 
the  aid  of  a  microscope. 


MICROMETRY  WITH  THE  SIMPLE  MICROSCOPE 
§  245.     With  a  simple  microscope  (i),  the  easiest  and  best  way 
is  to  use  dividers  and  then  with  the  simple  microscope  determine 
when  the  points  of  the  dividers 
exactly  include  the  object.     The 
spread  of  the  dividers  is  then 
obtained  as  above  (§  230-231). 
This  amount  will  be  the  actual 
size  of   the  object,  as  the  mi- 
croscope was  only  used  in  help- 
ing  to    see   when    the    divider 
points     exactly     enclosed     the 
object. 

(2)  One  may  put  the  object 
under  the  simple  microscope  and 
then,  as  in  determining  the 
power  (§  230),  measure  the  im- 
age at  the  standard  distance. 
If  the  size  of  the  image  so  meas- 
ured is  divided  by  the  magnifi- 
cation of  the  simple  microscope, 
the  quotient  gives  the  actual 
size  of  the  object.  One  might 
use  the  eikonometer  also 

(§  254). 

Use  a  fly's  wing  or  some  other 
object  of  about  that  size  and  try 
to  determine  the  width  in  the 
two  ways  described  above.  If 
all  the  work  is  accurately  done 
the  results  will  agree. 


FIG.  94.  FIELD  OF  THE  MICROSCOPE 
SHOWING  THE  LINES  AND  THE  RECORD- 
ING COMB  OF  THE  FILAR  MICROM- 
ETER (Fie.  93). 

C  The  recording  comb.  Each  tooth 
represents  a  complete  revolution  of  the 
drum  (fig.  93). 

//,  //     The  fixed  cross  lines. 

ml,  ml     The  movable  line. 

The  arrow  shows  that  the  movable 
line  can  be  moved  in  both  directions. 

O  Object,  the  full  movable  line  (ml) 
shows  it  at  one  edge  of  the  object  and 
the  broken  line  shows  it  at  the  other 
edge  of  the  object.  The  intervening 
teeth  of  the  comb  show  that  the  screw 
was  turned  two  whole  revolutions  and 
the  recording  drum  showed  90  divisions, 
making  two  and  nine  tenths  revolutions 
of  the  drum  to  carry  the  movable  line 
from  one  edge  of  the  object  to  the  other. 


1 50  MEASURING  WITH  THE  MICROSCOPE  [Cn.  V 

MICROMETRY    WITH    THE    COMPOUND    MICROSCOPE 

There  are  several  ways  of  varying  excellence  for  obtaining  the  size 
of  objects  with  the  compound  microscope,  the  method  with  the  ocular 
micrometer  (§  238)  being  most  accurate. 

§  246.  Unit  of  measure  in  micrometry.  —  Most  of  the  objects  measured  with 
the  compound  microscope,  and  many  of  those  in  physics  and  chemistry  are  smaller, 
often  much  smaller,  than  any  of  the  originally  named  divisions  of  the  meter. 
To  express  these  very  small  dimensions  in  common  or  in  decimal  fractions  of  a 
meter  or  millimeter  is  not  only  cumbersome,  but  liable  to  give  rise  to  errors,  con- 
sequently workers  in  microscopy,  in  physics  and  in  chemistry  have  sought  to  avoid 
the  difficulties  by  selecting  and  naming  as  units  such  small  divisions  of  the  meter 
that  the  minute  dimensions  can  be  expressed  as  whole  numbers. 

Up  to  the  present  three  such  special  units  have  been  designated  and  have  re- 
ceived the  sanction  and  use  of  the  highest  authorities.  They  are: 

1.  The  Micron  (symbol  /*).    This  is  the  one  millionth  of  a  meter  (o.ooo  ooi,  m.); 
one  thousandth  of  a  millimeter  (o.ooi  mm.);  ten  thousand  Angstrom  units  (ic,ooo 
A.U.);   one  thousand  millimicrons  (1000  mju). 

2.  The  Angstrom  Unit  (A.U.)  or  Tenthmeter  (io~10  m.).     It  is  the  one  ten 
billionth  of  a  meter  (o.ooo  ooo  ooo  i  m) ;  the  ten  thousandth  of  a  micron  (o.ooo  i  /i) ; 
the  one  tenth  of  a  millimicron  (o.i  m/x). 

3.  The  Millimicron  (mju).    This  is  the  one  billionth  of  a  meter  (o.ooo  ooo  ooi  m.) ; 
the  one  thousandth  of  a  micron  (o.ooi  ju);   ten  Angstrom  units  (10  A.U.). 

The  Micron  unit  (ju)  has  been  generally  adopted  in  microscopy,  and  is  widely 
used  for  minute  sizes  in  all  branches  of  science.  Harting  recommended  it  for  mi- 
croscopy in  1859,  but  he  named  it  micro-millimeter,  or  milli-millimeter,  and  gave 
as  a  symbol  mmm.  Since  the  definite  meaning  for  micro,  as  one  millionth  of  the 
unit  before  which  it  is  placed,  has  been  decided  on  by  metrologists,  micro-milli- 
meter should  mean  one  millionth  of  a  millimeter,  not  one  thousandth.  Harting' s 
Milli-millimeter  is  correct,  but  awkward.  Occasionally  one  meets  the  symbol  jiiju 
for  millimicron  (m/z).  /z/x  should  stand  for  the  millionth,  not  for  the  thousandth, 
of  a  micron. 

See  Jour.  Roy.  Micr.  Jour.  Soc.,  1888,  p.  502  Nature,  Vol.  XXXVII,  p.  388; 
Bit.  Bureau  Standards,  Vol.  VIII,  p.  540. 

§  247.  Micrometry  by  the  use  of  a  stage  micrometer  on  which  to 
mount  the  object.  —  In  this  method  the  object  is  mounted  on  a  mi- 
crometer and  then  put  under  the  microscope,  and  the  number  of 
spaces  covered  by  the  object  is  read  off  directly.  It  is  exactly  like 
putting  any  large  object  on  a  rule  and  seeing  how  many  spaces  of  the 
rule  it  covers.  The  defect  in  the  method  is  that  it  is  impossible  to 
properly  arrange  objects  on  the  micrometer.  Unless  the  objects 
are  circular  in  outline  they  are  liable  to  be  oblique  in  position,  and  in 
every  case  the  end  or  edges  of  the  object  may  be  in  the  middle  of  a 


CH.  V]  MEASURING    WITH    THE    MICROSCOPE  151 

space  instead  of  against  one  of  the  lines,  consequently  the  size  must 
be  estimated  or  guessed  at  rather  than  really  measured. 

§  248.  Micrometry  by  dividing  the  size  of  the  image  by  the  mag- 
nification of  the  microscope.  —  For  example,  employ  the  4  mm.  ob- 
jective, and  8x  or  lox  ocular.  For  measurement  use  a  preparation  of 
the  blood  corpuscles  of  the  frog,  necturus,  or  other  animal  with  large 
oval  corpuscles.  Obtain  the  size  of  the  image  of  the  long  and  short 
axes  of  three  corpuscles  with  the  camera  lucida  and  dividers,  exactly 
as  in  obtaining  the  magnification  of  the  microscope  (§  234).  Divide 
the  size  of  the  image  in  each  case 
by  the  magnification/,  and  the  result 
gives  the  actual  size  of  the  blood 
corpuscles.  Thus,  suppose  the  im- 
age of  the  long  axis  of  the  corpuscle 


is   1 8   mm.   and   the  magnification         FIG.  95.    BLOOD  PREPARATION 

of    the   microscope    400    diameters      WIT«  A  RING  AROUND  A  GROUP 

OF  CORPUSCLES. 
(§  228),  then  the  actual  length  of  this 

long  axis  of  the  corpuscle  is  18  mm.  -5-  400  =  0.045  mm.  or  45 /x  (§  231). 

As  the  same  three  blood  corpuscles  are  to  be  measured  in  three 
ways,  it  is  an  advantage  to  put  a  delicate  ring  around  a  group  of  three 
or  more  corpuscles,  and  make  a  sketch  of  the  whole  enclosed  group, 
marking  on  the  sketch  the  corpuscles  measured  (fig.  95).  The  differ- 
ent corpuscles  vary  considerably  in  size,  so  that  accurate  comparison 
of  different  methods  of  measurement  can  only  be  made  when  the  same 
corpuscles  are  measured  in  each  of  the  ways. 

§  249.  Micrometry  by  the  use  of  a  stage  micrometer  and  a  camera 
lucida.  —  Employ  the  same  object,  objective,  and  ocular  as  before. 
Put  the  camera  lucida  in  position,  and  with  a  lead  pencil  make  dots 
on  the  paper  at  the  limits  of  the  image  of  the  blood  corpuscles.  Meas- 
ure the  same  three  that  were  measured  in  §  248. 

Remove  the  object,  place  the  stage  micrometer  under  the  micro- 
scope, focus  well,  and  draw  the  lines  of  the  stage  micrometer  so  as  to 
include  the  dots  representing  the  limits  of  the  part  of  the  image  to  be 
measured.  As  the  value  of  the  spaces  on  the  stage  micrometer  is 
known,  the  size  of  the  object  is  determined  by  the  number  of  spaces 
of  the  micrometer  required  to  include  it. 


152  MEASURING    WITH    THE    MICROSCOPE  [CH.  V 

This  simply  enables  one  to  put  the  image  of  a  fine  rule  on  the  image 
of  a  microscopic  object.  It  is  theoretically  an  excellent  method,  and 
nearly  the  same  as  measuring  the  spread  of  the  dividers  with  a  simple 
microscope  (§  231). 

§  250.  Micrometry  with  the  ocular  micrometer  with  fixed  lines.  — 
Use  the  4  mm.  objective,  and  the  ocular  with  the  ocular  micrometer. 
For  object  use  the  same  corpuscles  as  in  §  248-249.  Make  sure  that 
all  the  conditions  are  exactly  as  when  the  valuation  was  determined; 
then  put  the  preparation  under  the  microscope  and  find  the  same  three 
red  corpuscles  that  were  measured  in  the  other  ways  (§  248). 

Count  the  divisions  on  the  ocular  micrometer  required  to  enclose 
or  measure  the  long  and  the  short  axis  of  each  of  the  corpuscles, 
multiply  the  number  of  spaces  in  both  cases  by  the  valuation  of  the 
ocular  micrometer,  and  the  results  will  represent  the  actual  length  of 
the  axes  of  the  corpuscles  in  each  case. 

The  same  corpuscle  is,  of  course,  of  the  same  actual  size,  when 
measured  in  each  of  the  three  ways,  so  that  if  the  methods  are 
correct  and  the  work  carefully  enough  done,  the  same  results  should 
be  obtained  by  each  method. 

§  251.  Micrometry  with  the  movable  scale  ocular  micrometer.  - 
Use  the  same  preparation  and  objective  as  before.  Arrange  the 
micrometer  ocular  so  that  the  long  axis  of  the  corpuscle  will  coincide 
with  the  cross  line  in  the  micrometer  scale  (fig.  91-92).  Get  one  end 
of  the  corpuscle  exactly  level  with  one  division  of  the  micrometer 
scale.  Note  the  position  of  the  drum,  and  then  rotate  it  until  the 
other  end  of  the  corpuscle  is  exactly  against  the  nearest  line  of  the 
micrometer.  Count  up  the  entire  intervals  required  and  the  partial 
interval  on  the  drum.  Suppose  it  requires  5  entire  and  0.60  inter- 
vals (see  explanation  of  fig.  92);  then  the  whole  corpuscle  must  be 
5.60  intervals  multiplied  by  4ft,  (§  242)  the  value  of  one  interval; 
5.6  x  4  =  22.4/x  . 

§  252.  Micrometry  with  the  filar  micrometer.  —  Use  the  same 
preparation  and  objective  as  before,  but  use  a  filar  micrometer.  Note 
how  many  graduations  on  the  recording  comb  and  drum  (fig.  93)  are 
required  to  measure  each  dimension  of  the  corpuscle,  and  multiply 
by  the  valuation  as  in  the  other  cases. 


CH.  V]  MEASURING    WITH    THE    MICROSCOPE  153 

The  advantage  of  the  filar  micrometer  is  that  the  valuation  of  one 
graduation  is  so  small  that  even  the  smallest  object  to  be  measured 
would  require  several  graduations  to  measure  it.  In  ocular  microm- 
eters with  fixed  lines,  small  objects  like  bacteria  might  not  fill  even 
one  space;  therefore  estimations,  not  measurements,  must  be  made. 
For  large  objects,  like  most  of  the  tissue  elements,  the  ocular  microm- 
eters with  fixed  lines  answer  very  well,  for  the  part  which  must  be 
estimated  is  relatively  small  and  the  chance  of  error  is  correspondingly 
small  (§  252a). 

§  252a.  There  are  three  ways  of  using  the  ocular  micrometer,  or  of  arriving 
at  the  size  of  the  objects  measured  with  it: 

(1)  By  finding  the  value  of  a  division  of  the  ocular  micrometer  for  each 
optical  combination  and  tube-length  used,  and  employing  this  valuation  as  a 
multiplier.     This  is  the  method  given  in  the  text,  and  the  one  most  frequently 
employed.     Thus,  suppose  with  a  given  optical  combination  and  tube-length 
it  required  five  divisions  on  the  ocular  micrometer  to  include  the  image  of  0.2 
millimeter  of  the  stage  micrometer,  then  obviously  one  space  on  the  ocular 
micrometer  would  include  1  or  0.2  or  0.04  mm.;    the  size  of  any  unknown 
object  under  the  microscope  would  be  obtained  by  multiplying  the  number 
of  the  divisions  on  the  ocular  micrometer  required  to  include  its  image  by  the 
value  of  one  space,  or  in  this  case  0.04  mm.     Suppose  some  object,  as  the  fly's 
wing,  required  15  spaces  of  the  ocular  micrometer  to  include  some  part  of  it, 
then  the  actual  size  of  this  part  of  the  wing  would  be  15  X  0.04  =  0.6  mm. 

(2)  By  finding  the  number  of  divisions  on  the  ocular  micrometer  required 
to  include  the  image  of  an  entire  millimeter  of  the  stage  micrometer,  and  using 
this  number  as  a  divisor.     This  number  is  also  sometim'es  called   the   ocular 
micrometer  ratio.     Taking  the  same  case  as  in  (i),  suppose  five  divisions  of 
the  ocular  micrometer  are  required  to  include  the  image  of  0.2  mm.,  on  the 
stage  micrometer,  then  evidently  it  would  require  5  -4-  0.2  =  25  divisions  on 
the  ocular  micrometer  to  include  a  whole  millimeter  on  the  stage  micrometer, 
and  the  number  of  divisions  of  the  ocular  micrometer  required  to  measure 
an  object  divided  by  25  would  give  the  actual  size  of  the  object  in  millimeters 
or  in  a  fraction  of  a  millimeter.     Thus,  suppose  it  required  15  divisions  of  the 
ocular  micrometer  to  include  the  image  of  some  part  of  the  fly's  wing,  the  actual 
size  of  the  part  included  would  be  15  -f-  25  =  f  or  0.6  mm.     This  method  is 
really  exactly  like  the  one  in  (i),  for  dividing  by  25  is  the  same  as  multiplying 
by  2*5-  or  0.04. 

(3)  By  having  the  ocular  micrometer  ruled  in  millimeters  and  divisions 
of  a  millimeter,  and  then  getting  the  size  of  the  real  image  in  millimeters.     In 
employing  this  method  a  stage  micrometer  is  used  as  object  and  the  size  of  the 
image  of  one  or  more  divisions  is  measured  by  the  ocular  micrometer,  thus: 
Suppose  the  stage  micrometer  is  ruled  o.i  and  o.oi  mm.  and  the  ocular  microm- 
eter is  ruled  in  millimeters  and  o.i  mm.     Taking  0.2  mm.  on  the  stage  microm- 
eter as  object,  as  in  the  other  cases,  suppose  it  requires  10  of  the  o.i  mm. 
spaces  or  i  mm.  to  measure  the  real  image,  then  the  real  image  must  be  magni- 
fied i.o  ^0.2  =  5  diameters,  that  is,  the  real  image  is  five  times  as  great  in 
length  as  the  object,  and  the  size  of  an  object  may  be  determined  by  putting 
it  under  the  microscope  and  getting  the  size  of  thVreal  image  in  millimeters 


154 


MEASURING    WITH    THE    MICROSCOPE 


[CH.  V 


with  the  ocular  micrometer  and  dividing  it  by  the  magnification  of  the  real 

image,  which  in  this  case  is  5  diameters. 

Use  the  fly's  wing  as  object,  as  in  the  other  cases, 
and  measure  the  image  of  the  same  part.  Suppose 
that  it  required  30  of  the  o.i  mm.  divisions  =  3  mm. 
to  include  the  image  of  the  part  measured,  then  evi- 
dently the  actual  size  of  the  part  measured  is  3  mm.  ^  5 
=  f  mm.,  or  0.6  mm.,  the  same  result  as  in  the  other 
cases.  See  also  §  253  on  the  eikonometer. 

In  comparing  these  methods  it  will  be  seen  that  in 
the  first  two  (i  and  2)  the  ocular  micrometer  may  be 
simply  ruled  with  equidistant  lines  without  regard  to 
the  absolute  size  in  millimeters  or  inches  of  the  spaces. 
In  the  last  method  the  ocular  micrometer  must  have 
its  spaces  some  known  division  of  a  millimeter  or  inch. 
In  the  first  two  methods  only  one  standard  of  measure 
is  required,  viz.,  the  stage  micrometer;  in  the  last 
method  two  standards  must  be  used,  viz.,  a  stage  mi- 
crometer and  an  an  ocular  micrometer. 


§  253.  Eikonometer  for  magnification  and 
micrometry.  —  The  eikonometer  is  something 
like  an  eye.  It  has  a  converging  lens  serving 
in  place  of  the  crystalline  lens  to  focus  the  rays 
from  the  eye-piece  of  the  compound  microscope, 
or  from  the  simple  microscope  upon  a  microm- 
eter scale,  the  scale  taking  the  place  of  the  retina 
in  the  eye  (fig.  77-78).  This  scale  is  ruled  in 
o.i  mm.  Above  the  scale  is  a  Ramsden  ocular 
of  25  mm.  equivalent  focus,  giving  a  magnifica- 
tion of  10.  The  eikonometer  scale  therefore  is 
a  millimeter  scale  when  seen  at  the  distance  of 
250  mm.  in  the  visual  field  of  the  normal  human 
eye,  and  it  enables  one  to  put  a  millimeter  scale 
on  the  image  of  any. object  studied. 

To  use  it  for  magnification  a  stage  micrometer 
is   put   under    the    microscope    and    carefully 
focused.     Then  the  eikonometer  is  put  in  place 
over  the  ocular.     The  microscopic  image  of  the 
stage  micrometer  and  the  scale  of  the  eikonom- 
eter will  then  appear  in  the  same  field  as  with  the  ordinary  ocular 
micrometer  (§  240).     The  two  sets  of  lines  should  be  made  parallel 


FIG.  96.     WRIGHT'S 
EIKONOMETER. 

(From  Sir  A.  E. 
Wright's  Principles 
of  Microscopy). 

o     Object. 

v  i    Virtual  image. 

ob     Objective. 

Microscope  Ocu- 
lar, the  objective, 
tube  and  ocular  of 
the  microscope. 

Eikonometer  The 
Ramsden  ocular  (Ro) 
magnifying  10  diam- 
eters, and  field  lens 
(//)  above  the  ocular 
of  the  microscope. 

es  The  real  image 
formed  at  the  dia- 
phragm of  the  eikon- 
ometer. 


CH.  V]  MEASURING    WITH    THE    MICROSCOPE  155 

(§  239~241)-  See  how  many  divisions  of  the  eikonometer  millimeter 
scale  are  required  to  measure  one  or  more  of  the  divisions  of  the  im- 
age of  the  stage  micrometer.  Suppose  it  requires  6  intervals  or  milli- 
meters of  the  eikonometer  scale  to  measure  the  image  of  0.03  mm.  on 
the  stage  micrometer.  The  size  of  the  object  is  then  0.03  mm.  and  of 
its  image  6  mm.  The  magnification  is  therefore  (§  228)  6  -=-  0.03  =  200. 

For  determining  the  magnification  of  a  simple  microscope  the  eiko- 
nometer is  placed  over  the  simple  microscope  as  it  was  over  the  ocular 
above.  With  this  instrument,  as  with  the  camera  lucida,  only  one 
eye  is  used  (fig.  81,  100). 

§  254.  Micrometry  with  the  eikonometer.  —  In  the  first  place  the 
magnification  of  the  microscope  must  be  determined  as  described 
in  the  preceding  section;  and  one  must  keep  in  mind  the  factors  which 
will  vary  the  magnification  (§  235).  The  object  to  be  measured  is 
put  under  the  microscope  and  focused  and  the  eikonometer  put  in 
position.  The  virtual  image  is  then  measured  in  millimeters  by  the 
scale  of  the  instrument.  The  size  of  this  virtual  image  is  then  divided 
by  the  magnification  and  the  result  will  be  the  actual  size  of  the 
object  as  in  §  248. 

For  example  suppose  the  long  axis  of  a  Necturus'  red  blood  corpuscle 
measures  9  mm.  on  the  eikonometer  scale.  If  the  magnification  of 
the  microscope  is  200,  as  found  above,  then  the  actual  length  of  the 
corpuscle  is  9  mm.  H-  200=  0.045  mm.,  or  45 p. 

§  255.  Micrometry  by  the  aid  of  tne  condenser  image  of  a  scale.  - 
Probably  every  one  is  all  too  familiar  with  the  cross  bars  of  the  window 
in  the  field  of  the  microscope.  This  is,  as  well  known,  a  real  image  of 
the  window  produced  by  the  condenser  at  the  level  of  the  object.  The 
possibility  of  projecting  a  real  image  at  the  level  of  the  object  is  taken 
advantage  of  for  purposes  of  micrometry  as  follows:  A  lantern  slide 
is  made  of  net  lines  (fig.  97)  or  of  any  parallel,  equidistant  lines.  The 
lantern  slide  is  then  set  up  exactly  10  cm.  or  some  other  exact  distance 
in  front  of  the  microscope.  A  good  light  from  the  window  or  from 
one  of  the  daylight  lanterns  (fig.  37-38)  must  traverse  the  lantern 
slide.  This  light  is  reflected  up  through  the  condenser  by  the  plane 
mirror.  The  condenser  will  form  a  real  image  of  the  network  or  par- 
allel lines  at  about  the  level  where  the  object  is  placed  on  the  slide. 


156 


MEASURING    WITH    THE    MICROSCOPE 


[CH.  V 


Tf  now  one  focuses  a  16  mm.  or  other  objective  upon  this  real  image, 
it  will  appear  very  clearly  in  the  field  of  the  microscope.  In  order  to 
utilize  the  image  for  micrometry  the  valuation  of  the  spaces  must  be 
determined  by  the  use  of  a  stage  micrometer  as  with  the  ocular  microm- 
eter (§  240).  Place  a  stage  micrometer  under  the  microscope  and 
focus  the  lines  sharply.  Then  with  the  screw  or  rack  of  the  substage 
condenser  focus  the  condenser  up  and  down  until  the  image  of  the 


FIG.  97. 


NET  SCALE  FOR  USE  IN  MICROMETRY  WITH  THE  CONDENSER 
IMAGE. 


lines  or  net  on  the  lantern  slide  are  also  sharp.  Arrange  the  stage 
micrometer  so  that  the  lines  are  parallel  with  the  lines  of  the  con- 
denser image.  Make  any  two  of  the  lines  coincide.  Count  the 
number  of  spaces  in  the  condenser  image  included  between  any 
two  of  the  lines  of  the  stage  micrometer,  and  divide  the  value  of 
the  space  in  the  stage  micrometer  by  the  number  of  spaces  of  the 
condenser  image  included,  and  the  quotient  will  represent  the  valua- 
tion of  the  spaces  of  the  condenser  image  in  millimeters.  For  ex- 
ample, suppose  the  stage  micrometer  is  ruled  in  o.i  mm.  and  that 
12  spaces  of  the  condenser  image  are  included  in  9  spaces  of  the  stage 
micrometer;  then  each  space  of  the  condenser  image  has  a  valuation 
of  0.9  mm.  -T-  12  =  0.075  mm. 

As  the  size  of  the  image  varies  with  the  distance  of  the  object  from 


CH.  V]  MEASURING    WITH    THE    MICROSCOPE  157 

the  center  of  the  condenser  (§  229),  if  the  object  (lantern  slide  of  the 
lines)  is  always  placed  exactly  the  same  distance  in  front  of  the  mi- 
croscope the  real  image  formed  by  the  condenser  will  be  of  the  same 
size,  and  hence  have  the  same  valuation  for  micrometry  regardless  of 
the  power  of  the  objective  or  the  length  of  tube  used.  It  is  a  very 
convenient  method  of  micrometry  for  all  coarser  objects,  but  not  exact 
enough  for  the  finer  objects.  A  movable  scale  or  filar  ocular  microm- 
eter should  be  used  for  the  most  exact  work. 

Example  of  an  actual  measurement  by  means  of  the  condenser 
image:  The  long  axis  of  a  red  corpuscle  of  Necturus  measured  0.6 1 
of  a  space  of  the  condenser  image.  As  each  space  represents  0.075 
mm.  the  length  of  the  corpuscle  is:  0.061  X  0.0.075  =  0.04575  mm- 
or  45. 75  ^  (see  Chamot,  pp.  155-157). 

§  256.  Remarks  on  micrometry.  —  In  using  adjustable  objectives 
(§31,  134)  the  magnification  of  the  objective  varies  with  the  position 
of  the  adjusting  collar,  being  greater  when  the  adjustment  is  closed, 
as  for  thick  cover-glasses,  than  when  open,  as  for  thin  ones.  This 
variation  in  the  magnification  of  the  objective  produces  a  corresponding 
change  in  the  magnification  of  the  entire  microscope  and  the  ocular 
micrometer  valuation;  therefore  it  is  necessary  to  determine  the 
magnification  and  ocular  micrometer  valuation  for  each  position  of 
the  adjusting  collar. 

While  the  principles  of  micrometry  are  simple,  it  is  very  difficult 
to  get  the  exact  size  of  microscopic  objects.  This  is  due  to  the  lack 
of  perfection  and  uniformity  of  micrometers  and  the  difficulty  of 
determining  the  exact  limits  of  the  object  to  be  measured.  Hence, 
all  microscopic  measurements  are  only  approximately  correct,  the 
error  lessening  with  the  increasing  perfection  of  the  apparatus  and 
the  skill  of  the  observer. 

A  difficulty  when  one  is  using  high  powers  is  the  width  of  the  lines 
of  the  micrometer.  If  the  micrometer  is  perfectly  accurate  half  the 
width  of  each  line  belongs  to  the  contiguous  spaces,  hence  one  should 
measure  the  image  of  the  space  from  the  centers  of  the  lines  bordering 
the  space,  or,  as  this  is  somewhat  difficult  in  using  the  ocular  mi- 
crometer, one  may  measure  from  the  inside  of  one  bordering  line  and 
from  the  outside  of  the  other,  that  is,  from  the  right  side  of  all  the 


158 


MEASURING    WITH    THE    MICROSCOPE 


[CH.  V 


lines,  or  from  the  left  side  of  all.  If  the  lines  are  of  equal  width  this 
is  as  accurate  as  measuring  from  the  center  of  the  lines.  Evidently  it 
would  not  be  right  to  measure  from  either  the  inside  or  the  outside 
of  both  lines  (fig.  90,  98). 

It  is  also  necessary  in  micrometry  to  use  an  objective  of  sufficient 
power  to  enable  one  to  see  all  the  details  of  an  object  with  great  dis- 
tinctness. The  necessity  of  using  sufficient  amplification  in  microm- 
etry has  been  especially  remarked  upon  by  Richardson,  Monthly 
Micr.  Jour.,  1874,  1875;  Rogers,  Proc.  Amer.  Soc.  Microscopists, 
1882,  p.  239;  Ewell,  North  Amer.  Pract.,  1890,  pp.  97,  173. 


Correct 


Correct 


FlG.  98. 


CORRECT   AND   INCORRECT    ARRANGEMENT  OF  THE 
OF  THE  STAGE  MICROMETER  LINES. 

(From   Chamot). 


Incorrect 
OCULAR 


AND 


The  fine  lines  are  those  of  the  ocular  micrometer  and  the  coarse  ones  of  the 
stage  micrometer  (compare  fig.  90). 

As  to  the  limit  of  accuracy  in  micrometry,  one  who  has  justly  earned 
the  right  to  speak  with  authority  expresses  himself  as  follows:  "I 
assume  that  0.2  JJL  is  the  limit  of  precision  in  microscopic  measures 
beyond  which  it  is  impossible  to  go  with  certainty."  W.  A.  Rogers, 
Proc.  Amer.  Soc.  Micrs.,  1883,  p.  198. 

In  comparing  the  methods  of  micrometry  with  the  compound 
microscope  given  above  (§  247-253),  the  one  given  in  §  247  is  im- 
practicable, that  given  in  §  252-3  is  open  to  the  objection  that  two 
standards  are  required,  —  the  stage  micrometer  and  the  steel  rule; 
it  is  open  to  the  further  objection  that  several  different  operations  are 
necessary,  each  operation  adding  to  the  probability  of  error.  Theo- 
retically the  method  given  in  §  249  is  good,  but  it  is  open  to  the  very 
serious  objection  in  practice  that  it  requires  so  many  operations  which 
are  especially  liable  to  introduce  errors.  The  method  that  experi- 
ence has  found  most  safe  and  expeditious,  and  applicable  to  all  objects, 


CH.  V]  MEASURING    WITH    THE    MICROSCOPE  159 

is  the  method  with  the  ocular  micrometer.  If  the  valuation  of  the 
ocular  micrometer  has  been  accurately  determined,  then  the  only 
difficulty  is  in  deciding  on  the  exact  limits  of  the  objects  to  be  measured 
and  so  arranging  the  ocular  micrometer  that  these  limits  are  enclosed 
by  some  divisions  of  the  micrometer.  Where  the  object  is  not  exactly 
included  by  whole  spaces  on  the  ocular  micrometer,  the  chance  of 
error  comes  in,  in  estimating  just  how  far  into  a  space  the  object 
reaches  on  the  side  not  in  contact  with  one  of  the  micrometer  lines. 
If  the  ocular  micrometer  has  some  quite  narrow  spaces,  and  others 
considerably  larger,  one  can  nearly  always  manage  to  exactly  include 
the  object  by  some  two  lines.  The  ocular  screw  micrometers  (fig.  91, 
94)  obviate  this  entirely,  as  the  cross  hair  or  lines  traverse  the  object 
or  its  real  image,  and  whether  this  distance  be  great  or  small  it  can  be 
read  off  on  the  graduated  wheel,  and  no  estimation  or  guess  work  is 
necessary. 

The  new  method  by  means  of  Wright's  eikonometer  (§  253-254) 
is  spoken  of  very  favorably  by  experts  who  have  employed  it. 

COLLATERAL  READING  FOR  CHAPTER  V 

Sir  A.  E.  Wright's  Principles  of  Microscopy.     Chamot,  Chemical  Microscopy. 

For  those  especially  interested  in  micrometry  in  its  relation  to  medical 
jurisprudence  the  following  are  recommended.  They  treat  the  subject  in  a 
practical  as  well  as  in  a  scientific  spirit.  The  papers  of  Prof.  Wm.  A.  Rogers 
on  micrometers  and  micrometry,  in  the  Amer.  Quar.  Micr.  Jour.,  Vol.  I.  pp.  97, 
208;  Proceedings  Amer.  Soc.  Microscopists,  1882,  1883,  1887.  Dr.  M.  D.  Ewell, 
Proc.  Amer.  Soc.  Micrs.,  1890;  The  Microscope,  1889,  pp.  43-45;  North  Amer. 
Pract.  1890,  pp.  97,  173.  Dr.  J.  J.  Woodward,  Amer.  Jour,  of  the  Med.  Sci., 
1875.  M.  C.  White,  Article  "Blood  Stains,"  Ref.  Hand-Book  Med.  Sciences, 
1885.  Medico-Legal  Journal,  Vol.  XII.  For  the  change  in  magnification  due 
to  a  change  in  the  adjustment  of  adjustable  objectives,  see  Jour.  Roy.  Micr. 
Soc.  1880,  p.  702;  Amer.  Monthly  Micr.  Jour.,  1880,  p.  67.  Carpenter-Dallinger, 
p.  270  and  end  of  §  196. 

If  one 'consults  the  medico-legal  Journals;  the  microscopical  journals,  the  Index 
Medicus,  and  the  Index  Catalog  of  the  library  of  the  Surgeon  General's  Office, 
under  Micometry,  Blood,  and  Jurisprudence,  he  can  get  on  track  of  the  main 
work  which  has  been  and  is  being  done. 


CHAPTER  VI 


DRAWING  WITH  THE  MICROSCOPE  AND  WITH  PROJECTION 
APPARATUS;   CLASS  DEMONSTRATIONS 

§  265.     Apparatus  and  material  for  Chapter  VI. 


1.  Microscope. 

2.  Abbe   and    Wollaston's   camera 
lucidas  (fig.  99-100). 

3.  Drawing   board    (fig.    101,    102, 
109). 

4.  Thumb   tacks  and   small  tacks 

(§  275)- 

5.  Pencils  (§  275). 

6.  Microscope  screen  (fig.  33). 

7.  Microscopic  preparations. 

8.  Small  arc  lamp  with  condenser 
(fig.  49). 

9.  Large  projection  apparatus   (fig. 
109-112). 

10.  45°  mirror  or  prism    (fig.    109, 
112-114). 

11.  Mazda    stereopticon    lamp    of 
250,  or  400  watts  (§  289,  294,  362).  ^ 

12.  Micrometer,      one-half      milli- 
meter, and  one  in  one-tenth  and  one- 
hundredth  millimeters  (fig.  80). 

13.  Mounted  letters. 

14.  Printed  letters  to  put  on  draw- 
ings (§  302). 

15.  Carbon  drawing  pencils  (§  290). 

16.  Graphite        drawing       pencils 
(§  288-290). 

17.  Water-proof    India    ink    (Hig- 
gin's  or  Weber's)  (§  288-290). 

1 8.  Crow  quill  pens  (§  288). 

19.  Right     line     pen     and     other 
drawing  instruments  (§  288). 


20.  Erasers. 

21.  Tracing  paper  (§  286). 

22.  Whatman's  hot-pressed  draw- 
ing paper  (§  291). 

23.  Reynold's  bristol-board  (§291). 

24.  Developing  photographic  paper 
(§  289-290). 

25.  Camera     obscura     or    photo- 
graphic   camera    and    material    for 
negatives  (fig.  107,  §  285-289). 

26.  Ruby  glass  (§  289). 

27.  Gihon's  opaque  and  fine  brush 
(§  290). 

28.  Metric  scale  (fig.  104). 

29.  T-square  and  triangles  (§  303). 

30.  Air  brush  (§  290). 

31.  Simple  microscopes  (§  306). 

32.  Demonstration    compound    mi- 
croscopes (§  307). 

33.  Traveling   microscope    (§  308). 

34.  Indicator  ocular  (§  309). 

35.  Markers  for  ringing  (§  310). 

36.  Projection  microscope  (§311). 

37.  Masking  paper  (§  i3i2a). 

38.  Objectives,   amplifiers,   oculars 


39.  Prism  or  45°  mirror  (§  315). 

40.  Hay  infusion  (§  211,  315). 

41.  Four-  window  daylight  lantern 
(§316). 

42.  Demonstration     table     for     8 
microscopes  (§  316). 


DRAWING 

§  266.  Methods  of  drawing.  —  There  are  five  principal  methods 
for  obtaining  drawings  in  general,  and  all  the  methods  are  applicable 
to  the  production  of  drawings  of  microscopic  objects: 

1 60 


CH.  VI]  DRAWING  WITH  A  CAMERA  LUCIDA  161 

(1)  Freehand  drawings.     This  is  the  simplest  method  if  one  has 
natural  ability  and  adequate  training,  for  one  only  needs  an  object, 
pencil,  pen  and  paper. 

(2)  Camera  lucida  drawings.     By  this  method  the  outlines  and 
proportions  can  be  accurately  traced  (§  268-275). 

(3)  Camera  obscura  drawings.     By  this  method  the  real  image 
obtained  in  a  photographic  camera  can  be  traced  (§  285). 

(4)  Projection  drawings.       In  this  method  real  images  like  those 
of  the  magic  lantern  and  projection  microscope  can  be  traced  directly 
upon  the  drawing  paper  (§  292). 

(5)  Line  drawings  on  blue  prints  and    on  the  back  of   photo- 
graphs (§  288-289). 

In  many  laboratories  all  the  methods  are  used,  sometimes  separately, 
but  more  often  combined. 

§  267.  Free-hand  drawings.  —  Microscopic  objects  may  be  drawn 
free-hand  directly  from  the  microscope,  but  in  this  way  a  picture 
giving  only  the  general  appearance  and  relations  of  parts  is  obtained. 
For  pictures  which  shall  have  all  the  parts  of  the  object  in  true  pro- 
portions and  relations,  it  is  necessary  to  obtain  an  exact  outline  of 
the  image  of  the  object,  and  to  locate  in  this  outline  all  the  principal 
details  of  structure.  It  is  then  possible  to  complete  the  picture  free- 
hand from  the  appearance  of  the  object  under  the  microscope. 

§  268.  Camera  lucida.  —  This  is  an  optical  apparatus  for  enabling 
one  to  see  objects  in  greatly  different  situations  as  if  in  one  field  of 
vision,  and  with  the  same  eye.  In  other  words,  it  is  an  optical  device 
for  superimposing  or  combining  two  fields  of  view  in  one  eye. 

As  applied  to  the  microscope,  it  causes  the  magnified  virtual  image 
of  the  object  under  the  microscope  to  appear  as  if  projected  upon  the 
table  or  drawing  board,  where  it  is  visible  with  the  drawing  paper, 
pencil,  dividers,  etc.,  by  the  same  eye,  and  in  the  same  field  of  vision. 
The  microscopic  image  appears  like  a  picture  on  the  drawing  paper 
(see  §  27ia).  This  is  accomplished  in  two  distinct  ways: 

(i)  By  a  camera  lucida  reflecting  the  rays  from  the  microscope 
so  that  their  direction  when  they  reach  the  eye  coincides  with  that 
of  the  rays  from  the  drawing  paper,  pencil,  etc.  In  some  of  the 
camera  lucidas  from  this  group  (Wollaston's,  fig.  99),  the  rays  are 


162 


DRAWING   WITH  A   CAMERA  LUCIDA 


[CH.  VI 


reflected  twice,  and  the  image  appears  as  when  looking  directly  into 
the  microscope.  In  others  the  rays  are  reflected  but  once,  and  the 
image  has  the  inversion  produced  by  a  plane  mirror.  For  drawing 
purposes  this  inversion  is  a  great  objection,  as  it  is  necessary  to 
^  similarly  invert  all  the  details 

added  free-hand. 

(2)  By  a  camera  lucida  re- 
flecting the  rays  of  light  from 
the  drawing  paper,  etc.  so 
that  their  direction  when  they 
reach  the  eye  coincides  with 
the  direction  of  the  rays  from 
the  microscope  (fig.  100).  In 
all  of  the  camera  lucidas  of 
this  group,  the  rays  from  the 
paper  are  twice  reflected  and 
no  inversion  appears. 

The  better  forms  of  camera 
lucidas  (Wollaston's,  Gru- 


FIG.  99.    WOLLASTON'S  CAMERA  LUCIDA. 

Axis    The  optic  axis  of  the  microscope. 

Ocular    The  upper  end  of  the  ocular. 

A,  B  Two  rays  outside  the  axis  to  show 
that  they  cross  twice  and  hence  have  the 
same  relative  position  as  when  they  emerge 
from  the  ocular. 

Camera  lucida  The  quadrangular  piece  of 
glass  giving  -the  double  internal  reflection  to 
change  the  direction  of  the  axial  ray  90°. 

CD,  A  B  The  virtual  image,  drawing  paper 
and  pencil  partly  overlapping.  Where  they 
overlap  the  appearance  is  that  of  one  field. 


now's,  Abbe's,  etc.)  may  be 
used  for  drawing  both  with 
low  and  with  high  powers. 
Some  require  the  microscope 
to  be  inclined  (fig.  99)  while 
others  are  designed  to  be  used 
on  the  microscope  in  a  vertical 
position.  As  in  biological 
work,  it  is  often  necessary  to 


have  the  microscope  vertical, 
the  form  for  a  vertical  microscope  is  to  be  preferred  (see  fig.  100). 

§  269.  Avoidance  of  distortion.  —  In  order  that  the  picture 
drawn  by  the  aid  of  a  camera  lucida  may  not  be  distorted,  it  is  neces- 
sary that  the  axial  ray  from  the  image  on  the  drawing  surface  shall 
be  at  right  angles  to  the  drawing  surface  (fig.  99,  101). 

§  270.  Wollaston's  camera  lucida.  —  This  is  a  quadrangular 
prism  of  glass  put  in  the  path  of  the  rays  from  the  microscope,  and 


CH.  VI]  DRAWING  WITH  A  CAMERA  LUCIDA  163 

it  serves  to  change  the  direction  of  the  axial  ray  90  degrees.  In  using 
it  the  microscope  is  made  horizontal,  and  the  rays  from  the  microscope 
enter  one-half  of  the  pupil,  while  rays  from  the  drawing  surface  enter 
the  other  half  of  the  pupil.  As  seen  in  fig.  99,  the  fields  partly  overlap, 
and  where  they  do  so  overlap,  pencil  or  dividers  and  microscopic 
image  can  be  seen  together. 

In  drawing  or  using  the  dividers  with  the  Wollaston  camera  lucida 
it  is  necessary  to  have  the  field  of  the  microscope  and  the  drawing 
surface  about  equally  lighted.  If  the  drawing  surface  is  too  bril- 
liantly lighted  the  pencil  or  dividers  may  be  seen  very  clearly,  but 
the  microscopic  image  will  be  obscure.  On  the  other  hand,  if  the 
field  of  the  microscope  has  too  much  light  the  microscopic  image  will 
be  very  definite,  but  the  pencil  or  dividers  will  not  be  clearly  visible. 
It  is  necessary,  as  with  the  Abbe  camera  lucida  (§271),  to  have  the 
Wollaston  prism  properly  arranged  with  reference  to  the  axis  of  the 
microscope  and  the  eye-point.  If  it  is  not,  one  will  be  unable  to  see 
the  image  well,  and  may  be  entirely  unable  to  see  the  pencil  and  the 
image  at  the  same  time.  Again,  as  rays  from  the  microscope  and 
from  the  drawing  surface  must  enter  independent  parts  of  the  pupil 
of  the  same  eye,  one  must  hold  the  eye  so  that  the  pupil  is  partly  over 
the  camera  lucida  and  partly  over  the  drawing  surface.  One  can 
tell  the  proper  position  by  trial.  This  is  not  a  very  satisfactory 
camera  to  draw  with,  but  it  is  a  very  good  form  to  measure  the  ver- 
tical distance  of  250  mm.  at  which  the  drawing  surface  should  be 
placed  when  determining  magnification  (fig.  85). 

§  271.  Abbe  camera  lucida.  —  This  consists  of  a  cube  of  glass 
cut  into  two  triangular  prisms  and  silvered  on  the  cut  surface  of  the 
upper  one.  A  small  oval  hole  is  then  cut  out  of  the  center  of  the 
silvered  surface  and  the  two  prisms  are  cemented  together  in  the  form 
of  the  original  cube  with  a  perforated  45  degree  mirror  within  it 
(fig.  100-101).  The  upper  surface  of  the  cube  is  covered  by  a  per- 
forated metal  plate.  This  cube  is  placed  over  the  ocular  in  such  a 
way  that  the  light  from  the  microscope  passes  through  the  hole  in 
the  silvered  face  and  thence  directly  to  the  eye.  Light  from  the 
drawing  surface  is  reflected  by  the  mirror  to  the  silvered  surface  of 
the  prism  and  reflected  by  this  surface  to  the  eye  in  company  with 


164 


DRAWING  WITH  A   CAMERA  LUCIDA 


[CH.  VI 


the  rays  from  the  microscope,  so  that  the  two  fields  appear  as  one,  and 
the  image  is  seen  as  if  on  the  drawing  surface  (fig.  100-102,  §  27ia). 


FIG.  ioo.     DIAGRAM  OF  ABBE'S  CAMERA  LUCIDA  WITH  A  VERTICAL  MICROSCOPE. 

Axis,  Axis  The  axial  ray  of  the  microscope  and  from  the  field  of  the  drawing 
surface. 

Ocular    The  upper  part  of  the  microscope  ocular. 

Mirror  The  mirror  of  the  camera  lucida  reflecting  the  rays  from  the  drawing 
surface  at  right  angles  to  the  axis. 

P,  P    The  drawing  pencil  in  the  field,  and  the  prism  of  the  camera  lucida. 

Q    The  quadrant  attached  to  the  mirror  to  give  the  angle. 

G    Smoked  glass. 

a  b  The  silvered  surface  in  the  prism  with  a  hole  made  in  the  center  for  the 
light  to  pass  upward  from  the  microscope.  The  silvered  part  reflects  the  rays  from 
the  drawing  surface. 

The  geometrical  figure  at  the  left  gives  the  angles  when  a  45°  mirror  is  used. 

§  271a.  For  some  persons  the  image  and  the  drawing  surface,  pencil,  etc.,  do 
not  appear  on  the  drawing  board  as  stated  above,  but  under  the  microscope,  ac- 
cording to  the  general  principle  that  "objects  appear  in  space  where  they  could 
be  touched  along  a  perpendicular  to  the  retinal  surface  stimulated,"  —  that  is,  in 


CH.  VI]  DRAWING  WITH  A  CAMERA  LUCID  A  165 

the  line  of  rays  entering  the  eye.  This  is  always  the  case  with  the  Wollaston  camera 
lucida.  The  explanation  of  the  apparent  location  of  the  image,  etc.,  on  the  draw- 
ing board  with  the  Abbe  camera  lucida  is  that  the  attention  is  concentrated  upon 
the  drawing  surface  rather  than  upon  the  object  under  the  microscope.  With 
some  observers  it  is  possible  to  make  the  image  appear  under  the  microscope  or 
on  the  drawing  surface  at  will  by  concentrating  the  attention  on  one  position  or 
the  other.  (Dr.  W.  B.  Pillsbury). 

§  272.  Arrangement  of  the  camera  lucida  prism.  —  In  placing 
this  camera  lucida  over  the  ocular  for  drawing  or  the  determination 
of  magnification,  the  center  of  the  hole  in  the  silvered  surface  is 
placed  in  the  optic  axis  of  the  microscope.  This  is  done  by  properly 
arranging  the  centering  screws  that  clamp  the  camera  to  the  micro- 
scope tube  or  ocular.  The  prism  must  not  only  be  centered  to  the 
axis  of  the  microscope,  but  it  must  be  at  the  right  level  or  more  or 
less  of  the  field  will  be  cut  off.  In  all  the  good  modern  forms  of 
this  camera  lucida  it  is  fastened  to  the  tube  of  the  microscope  by  a 
clamp  which  enables  one  to  raise  or  lower  it  so  that  it  may  be  at 
the  right  position  with  reference  to  the  eye-point  of  the  ocular  being 
used  (§  57). 

One  can  determine  when  the  camera  is  in  a  proper  position  by  look- 
ing into  the  microscope  through  it.  If  the  field  of  the  microscope 
appears  as  a  circle  and  of  about  the  same  size  as  without  the  camera 
lucida,  then  the  prism  is  in  a  proper  position.  If  one  side  of  the 
field  is  dark,  then  the  prism  is  to  one  side  of  the  center;  if  the  field 
is  considerably  smaller  than  when  the  prism  is  turned  off  the  ocular, 
it  indicates  that  it  is  not  at  the  correct  level,  i.e.,  it  is  above  or  too 
far  below  the  eye-point. 

§  273.  Arrangement  of  the  mirror  and  the  drawing  surface.  — 
The  Abbe  camera  lucida  was  designed  for  use  writh  a  vertical  micro- 
scope (fig.  100).  On  a  vertical  microscope  if  the  mirror  is  set  at  an 
angle  of  45°,  the  axial  ray  is  at  right  angles  with  the  table  top  or 
drawing  board  which  is  horizontal,  and  a  drawing  made  under  these 
conditions  is  in  true  proportion  and  not  distorted.  The  stage  of 
most  microscopes,  however,  extends  out  so  far  at  the  sides  that  with 
a  45°  mirror  the  image  appears  in  part  on  the  stage  of  the  microscope. 
In  order  to  avoid  this  the  mirror  may  be  depressed  to  some  point 
below  45°,  say  at  40°  or  35°  (fig.  101).  But  as  the  axial  ray  from 


i66 


DRAWING  WITH  A  CAMERA  LUCIDA 


[CH.  VI 


B 


the  mirror  to  the  prism  must  still  be  reflected  horizontally,  it  follows 
that  the  axial  ray  no  longer  forms  an  angle  of  90°  with  the  drawing 

surface,  but  a  greater 
angle.  If  the  mirror  is 
depressed  to  35°,  then 
the  axial  ray  makes 
an  angle  of  110°  with 
a  horizontal  drawing 
surface  (fig.  101  B) :  To 
make  the  angle  90° 
again,  so  that  there 
shall  be  no  distortion, 
the  drawing  board 
must  be  raised  toward 
the  microscope  20°. 
The  general  rule  is  to 
raise  the  drawing  board 
twice  as  many  degrees 
toward  the  microscope 
as  the  mirror  is  de- 
pressed below  45°. 
Practically  the  field 
for  drawing  can  al- 
ways be  made  free  of 
the  stage  of  the  micro- 


FIG.  101.  DIAGRAM  OF  THE  ABBE  CAMERA  LU- 
CIDA WITH  THE  DRAWING  SURFACE  ELEVATED  TO 
MAKE  THE  Axis  PERPENDICULAR  WITH  DEPRESSED 
MIRROR. 

A,  Axis,  Axis  The  axial  ray  from  the  microscope 
and  from  the  drawing  surface. 

Ocular    The  upper  part  of  the  microscopic  ocular. 

Mirror  The  minor.of  the  camera  lucida;  it  is  de- 
pressed from  45°  to  35°  to  make  the  axis  from  the 
drawing  surface  perpendicular  to  the  axis  of  the  micro- 
scope. 

A  — B  The  drawing  surface  elevated  20°;  that  is, 
twice  as  many  as  the  mirror  is  depressed  below  45°. 

W    Wedge  under  the  drawing  board. 

P,  P  The  drawing  pencil  and  the  prism  of  the 
camera  lucida. 

Q    Quadrant  of  the  mirror. 

B  Geometrical  figure  to  show  why  the  drawing 
board  must  be  raised  twice  as  many  degrees  as  the 
mirror  is  depressed  to  keep  the  axial  ray  perpendicu- 
lar to  the  drawing  surface. 


scope,  at  45°,  at  40°, 
or  at  35°.  In  the  first 
case  (45°  mirror) 


the 

drawing  surface  should 
be  horizontal,  in  the 
second  case  (40°  mir- 
ror) the  drawing  sur- 


face should  be  elevated 

10°,  and  in  the  third  case  (35°  mirror)  the  drawing  board  should  be 
elevated  20°  toward  the  microscope.  Furthermore  it  is  necessary 
in  using  an  elevated  drawing  board  to  have  the  mirror  bar  of  the 


CH.  VI]  DRAWING  WITH  A  CAMERA  LUCIDA  167 

camera  lucida  project  directly  laterally  so  that  the  edges  of  the  mirror 
are  in  planes  parallel  with  the  edges  of  the  drawing  board;  otherwise 
there  will  be  front  to  back  distortion,  although  the  elevation  of  the 
drawing  board  avoids  right  to  left  distortion.  If  one  has  a  microm- 
eter ruled  in  squares  (net  micrometer)  (fig.  65,  97),  the  distortion 
produced  by  not  having  the  axial  ray  at  right  angles  with  the  drawing 
surface  may  be  very  strikingly  shown.  For  example,  set  the  mirror 
at  35°  and  use  a  horizontal  drawing  board.  With  a  pencil  make 
dots  at  the  corners  of  some  of  the  squares,  and  then  with  a  straight 
edge  connect  the  dots.  The  figures  will  be  considerably  longer  from 
right  to  left  than  from  front  to  back.  Circles  in  the  object  appear 
as  ellipses  in  the  drawings,  the  major  axis  being  from  right  to  left. 

The  angle  of  the  mirror  may  be  determined  with  a  protractor, 
but  that  is  troublesome.  It  is  much  more  satisfactory  to  have  a 
quadrant  attached  to  the  mirror  and  an  indicator  on  the  projecting 
arm  of  the  mirror.  If  the  quadrant  is  graduated  throughout  its 
entire  extent,  or  preferably  at  three  points,  45°,  40°  and  35°,  one  can 
set  the  mirror  at  a  known  angle  in  a  moment;  then  the  drawing  board 
can  be  hinged  and  the  elevation  of  10°  and  20°  determined  with  a 
protractor.  The  drawing  board  is  very  conveniently  held  up  by  a 
broad  wedge.  By  marking  the  position  of  the  wedge  for  10°  and 
20°  the  protractor  need  be  used  but  once;  then  the  wedge  may  be 
put  into  position  at  any  time  for  the  proper  elevation. 

§  274.  Abbe  camera  and  inclined  microscope.  —  It  is  very  fati- 
guing to  draw  continuously  with  a  vertical  microscope,  and  many 
mounted  objects  admit  of  an  inclination  of  the  microscope,  when  one 
can  sit  and  work  in  a  more  comfortable  position.  The  Abbe  camera 
is  perfectly  adapted  to  use  with  an  inclined  as  with  a  vertical  micro- 
scope. All  that  is  requisite  is  to  be  sure  that  the  fundamental  law 
is  observed  regarding  the  axial  ray  of  the  image  and  the  drawing 
surface,  viz.  that  they  should  be  at  right  angles.  This  is  very  easily 
accomplished  as  follows:  The  drawing  board  is  raised  toward  the 
microscope  twice  as  many  degrees  as  the  mirror  is  depressed  below 
45°  (§  273)  5  then  it  is  raised  exactly  as  many  degrees  as  the  micro- 
scope is  inclined,  and  in  the  same  direction,  that  is,  so  that  the  end 
of  the  drawing  board  shall  be  in  a  plane  parallel  with  the  stage  of 


i68 


DRAWING  WITH  A  CAMERA  LUCIDA 


[CH.  VI 


the  microscope.     The  mirror  must  have  its  edges  in  planes  parallel 
with  the  edges  of  the  drawing  board  also  (fig.  102). 

§  275.  Drawing  with  the  Abbe  camera  lucida.  —  (i)  The  light 
from  the  microscope  and  from  the  drawing  surface  should  be  of 
nearly  equal  intensity,  so  that  the  image  and  the  drawing  pencil  can 


FIG.  102.    BERNHARD'S  DRAWING  BOARD  FOR  THE  ABBE  CAMERA  LUCIDA. 
(From  the  Catalogue  of  Zeiss). 

This  drawing  board  can  be  elevated  and  tipped;   it  can  also  be  inclined,  carrying 
the  microscope  with  it. 

be  seen  with  about  equal  distinctness.  This  may  be  accomplished 
with  very  low  powers  (16  mm.  and  lower  objectives)  by  covering  the 
mirror  of  the  microscope  with  white  paper  when  transparent  objects 
are  to  be  drawn.  For  high  powers  it  is  best  to  use  a  substage  con- 
denser. Often  the  light  may  be  balanced  by  using  a  larger  or  smaller 
opening  in  the  diaphragm.  One  can  tell  which  field  is  excessively 
illuminated,  for  it  is  the  one  in  which  objects  are  most  distinctly  seen. 


CH.  VI]  DRAWING  WITH  A  CAMERA  LUCID  A  169 

If  it  is  the  microscopic,  then  the  image  of  the  microscopic  object  is 
very  distinct  and  the  pencil  is  invisible  or  very  indistinct.  If  the 
drawing  surface  is  too  brilliantly  lighted  the  pencil  can  be  seen  clearly, 
but  the  microscopic  image  is  obscure. 

When  opaque  objects,  that  is,  objects  which  must  be  lighted  with 
reflected  light  (fig.  21,  34),  like  dark  colored  insects,  etc.,  are  to 
be  drawn,  the  light  must  usually  be  concentrated  upon  the  object  in 
some  way.  The  microscope  may  be  placed  in  a  very  strong  light 
and  the  drawing  board  shaded,  or  the  light  may  be  concentrated  upon 
the  object  by  means  of  a  concave  mirror,  or  a  bull's  eye  condenser 
or  the  small  arc  lamp  (fig.  49)  may  be  used. 

If  the  drawing  surface  is  too  brilliantly  illuminated,  it  may  be 
shaded  by  placing  a  book  or  a  ground-glass  screen  between  it  and 
the  window,  also  by  putting  one  or  more  smoked  glasses  in  the  path 
of  the  rays  from  the  mirror  (fig.  100).  If  the  light  in  the  microscope 
is  too  intense,  it  ma^  be  lessened  by  using  white  paper  over  the  mirror, 
or  by  a  ground-glass  screen  between  the  microscope  mirror  and  the 
source  of  light  (Piersol,  American  Monthly  Microscopical  Journal, 
1888,  p.  103).  It  is  also  an  excellent  plan  to  blacken  the  end  of  the 
drawing  pencil  with  carbon  ink.  Sometimes  it  is  easier  to  draw  on  a 
black  surface,  using  a  white  pencil  or  style.  The  carbon  paper  used 
in  manifolding  letters,  etc.,  may  be  used,  or  ordinary  black  paper 
may  be  lightly  rubbed  on  one  side  with  a  moderately  soft  lead  pencil. 
Place  the  black  paper  over  white  paper  and  trace  the  outlines  with 
a  pointed  style  of  ivory  or  bone.  A  corresponding  dark  line  will 
appear  on  the  white  paper  beneath  (Jour.  Roy.  Micr.  Soc.,  1883, 

P-  423)- 

(i)  It  is  desirable  to  have  the  drawing  paper  fastened  with  thumb 
tacks,  or  in  some  other  way.  (2)  The  lines  made  while  using  the 
camera  lucida  should  be  very  light,  as  they  are  liable  to  be  irregular. 
(3)  Only  outlines  are  drawn  and  parts  located  with  a  camera  lucida. 
Details  are  put  in  free-hand.  (4)  It  is  sometimes  desirable  to  draw 
the  outline  of  an  object  with  a  moderate  power  and  add  the  details 
with  a  higher  power.  If  this  is  done  it  should  always  be  clearly 
stated.  It  is  advisable  to  do  this  only  with  objects  in  which  the 
same  structure  is  many  times  duplicated,  as  a  nerve  or  a  muscle. 


170  SCALE  OF  DRAWINGS  [CH.  VI 

In  such  an  object  all  the  different  structures  can  be  shown,  and  by 
omitting  some  of  the  fibers  the  others  may  be  made  plainer  with- 
out undesirable  enlargement  of  the  entire  figure.  (5)  If  a  drawing 
of  a  given  size  is  desired  and  it  cannot  be  obtained  by  any 
combination  of  oculars,  objectives,  and  lengths  of  the  tube  of 
the  microscope,  the  distance  between  the  camera  lucida  and  the 
table  may  be  increased  or  diminished  until  the  image  is  of  the  desired 
size.  This  distance  is  easily  changed  by  the  use  of  a  book  or  a  block, 
but  more  conveniently  if  one  has  a  drawing  board  with  adjustable 
drawing  surface  like  that  shown  in  fig.  102.  (6)  It  is  of  advantage 
to  have  the  camera  lucida  hinged  so  that  the  prism  may  be 
turned  off  the  ocular  for  a  moment's  glance  at  the  preparation, 
and  then  returned  in  place  without  the  necessity  of  loosening 
screws  and  readjusting  the  camera.  This  form  is  now  made 
by  several  opticians,  and  many  of  them  add  graduations  so  that 
the  angle  of  the  mirror  is  readily  seen. 

§  276.  Scale  of  drawings.  —  The  scale  should  be  given  for  every 
drawing  (fig.  103).  Sometimes  the  drawing  is  larger  than  the  object, 
as  with  microscopic  specimens,  and  sometimes  it  is  of  the  same  size 
or  much  smaller,  as  in  drawing  large  objects. 

In  getting  the  scale  at  which  an  object  is  drawn  with  the  microscope 
or  projection  microscope,  the  object  is  removed  and  a  micrometer 
in  half  millimeters  (fig.  65)  for  low  powers  and  one  in  tenths  and 
hundredths  of  a  millimeter  (fig.  80)  for  high  powers  is  put  in  place 
of  the  specimen.  The  image  of  the  micrometer  lines  and  spaces  will 
be  of  the  same  enlargement  as  the  drawing,  provided  nothing  has 
been  changed  except  the  micrometer  for  the  object.  If  now  a  few  of 
the  lines  of  the  micrometer  image  (fig.  80,  103)  are  traced  at  one 
corner  of  the  drawing  paper  and  their  actual  value  given,  the  enlarge- 
ment can  be  determined  accurately  as  follows:  Suppose  the  mi- 
crometer spaces  are  tenth  millimeters,  and  the  image  of  the  spaces 
measures  2  millimeters,  the  enlargement  must  be  the  size  of  the 
image  divided  by  the  size  of  the  object  or  2  -=-  o.i  =  20,  that  is,  the 
image  is  20  times  the  size  of  the  object. 

In  using  the  photographic  camera  for  negatives  or  for  tracing, 
if  the  metric  scale  (fig.  104)  is  put  with  the  object  its  image  will 


CH.  VlH  SCALE  OF  DRAWINGS  171 

appear  with  the  image  in  the  negative  or  in  the  tracing  and  the  en- 
largement or  reduction  can  be  found  as  above.  Suppose  the  image 
of  the  10  cm.  scale  on  the  negative  or  in  the  tracing  is  2  cm.  long, 
obviously  the  picture  must  be  2  cm.  -r-  10  =  -?$  or  y,  that  is,  the 
picture  is  only  one-fifth  the  size  of  the  object. 

For  any  form  of  projection  apparatus  (fig.  109-114),  the  magic 
lantern  or  projection  microscope,  after  the  image  is  traced,  the  object 
is  removed  and  a  micrometer  in  half  millimeters  for  the  magic  lantern 
and  low  powers  of  the  microscope  is  put  in  place  of  the  object  and 
the  image  of  the  scale  projected  upon  the  drawing  paper.  Suppose 
the  image  of  one  of  the  micrometer  half  millimeter  spaces  measures 
15  millimeters,  then  the  scale  of  the  drawing 
must  be  30  (i.e.,  15  -5-  ^  =  30). 

If  one  is  drawing  from  the  projected  image 
of  a  negative  or  lantern  slide  it  is  necessary  iM»  mm- 

to  know  the  scale  at  which  the  negative  or  FIG.  103.  MAGNIFIED 
slide  was  made  as  well  as  the  scale  at  which  o? 


the  drawing  from  the  projected  negative  or     INDICATING  THE  SCALE 
i-j    •    *    •  i        T-«  i     •£  AI_          i        AT  WHICH  A  DRAWING 

slide  is  being  made,     r  or  example,  it  the  scale     WAS 


of  the  negative  is  50  times  the  size  of  the  ob- 

ject, and  the  drawing  is  10  times  the  size  of  the  negative,  the  final 

drawing  must  be  10  X  50  =  500  times  the  size  of  the  original  object. 

If  on  the  other  hand  the  negative  is  yV  the  size  of  the  original 
object  and  the  drawing  is  5  times  the  size  of  the  negative,  the  final 
drawing  will  be  the  size  of  the  negative  (iV  the  original)  multiplied 
by  the  magnification  (in  this  case  5)  which  is  ro  X  5  =  -fy  or  y. 
That  is,  the  drawing  is  one-half  the  size  of  the  original  object. 

For  the  projection  microscope  with  powers  from  40  to  16  mm.  a 
micrometer  in  y  mm.  is  good.  For  powers  above  16  mm.  it  is  better 
to  use  a  micrometer  in  o.i  mm.  and  o.oi  mm.  (fig.  80). 

After  the  drawing  has  been  made,  remove  the  specimen  and  put 
the  micrometer  under  the  microscope  and  draw  a  few  spaces  of  the 
micrometer  image  (fig.  103)  giving  the  actual  value  of  the  spaces; 
then  one  can  compute  the  enlargement  of  the  drawing  by  measuring 
the  image  spaces  and  dividing  by  the  actual  value.  For  example, 
suppose  the  image  of  one  of  the  o.i  mm.  spaces  measures  on  the 


172  AVOIDANCE   OF   INVERSION;    ERECT   IMAGES        [Cn.  VI 

drawing  4  cm.  or  40  mm.,  the  scale  of  the  drawing  or  its  magnification 
is  40.  -r-  o.i   =  400. 

§  276a.  For  diagrams  and  other  large  objects  a  very  serviceable  micrometer 
can  be  made  by  using  the  10  cm.  metric  rule  (fig.  104)  as  object  and  making  a 
negative  of  it  on  a  lantern  slide  exactly  natural  size  or  half  natural  size. 


0 


10  CENTIMETER  RULE. 
The  upper  edge  is  in  millimeters,  the  lower  in  centimeters. 


THE  METRIC  SYSTEM. 

UNITS.  The  most  commonly  used  divisions  ami  multiples. 

{    Centimeter  (cm),    o.oi    Meter;  Millimeter  (mm.),  o.ooi  Meter :   Micron  ({i>,   o.ooi 
THE  METER  FOR  A  Millimeter;  the  Micron  is  the  unit  in  Micrometry. 

LENGTH          f    Kilometer,  1000  Meters;  used  in  measuring  roads  and  other  long  distances. 
THE  GRAM  FOR    j    Milligram  (mg.),  o.ooi  Gram. 

WEIGHT          I    Kilogram,  1000   Grams,  used  for  ordinary  masses,    like  groceries,  etc. 
THE  LITER  FOR   j    Cubic  Centimeter,  (cc.),  o.ooi  Liter.      This  is  more  common  than  the  correct 
CAPACITY.          I  form,  Milliliter. 

Divisions  of  the  Units  are  indicated  by  the  Latin  prefixes;  deci.  o.i ;  cent/,   o.oi ;  ntilli,  o.ooi ; 
micro,  one  millionth  (o.oooooi)  of  any  unit. 

Multiples  are  designated  by  the  Greek    prefixes;  dcka,    10   times;  hecto,    100   times;  kiln,    1000 
times;  myria,  10,000  times;  Mega,  one  million  (1,000,000)  times  any  unit. 

FIG.  104.     METRIC  SCALE  AND  SUMMARY  OF  THK  METRIC  SYSTEM. 

AVOIDANCE  OF  INVERSION 

§  277.  It  is  desirable  to  make  drawings  like  the  object  without 
any  inversion  whatsoever,  provided  the  object  has  rights  and  lefts, 
etc.  For  structural  detail  like  cells,  etc.,  it  makes  no  difference 
whether  the  image  is  erect  or  not,  but  with  symmetrical  organs  and 
animals  it  is  very  confusing  to  have  the  parts  inverted  in  the  drawing. 
For  example,  it  is  unsatisfactory  to  have  the  liver  shown  as  if  on  the 
left  side  and  the  heart  on  the  right  side. 

In  order  to  avoid  inversions,  it  is  necessary  to  know  what  inver- 
sions are  produced  by  the  different  optical  appliances  used^  to  assist 
in  drawing.  Then  one  can  so  arrange  the  object  that  the  image 


CH.  VI]        AVOIDANCE   OF   INVERSION;    ERECT  IMAGES 


173 


will  be  exactly  like  the  object.  It  is  believed  that  the  following 
directions  will  enable  the  worker  to  so  arrange  his  specimen  and  the 
apparatus  that  erect  images  may  be  produced  without  undue  effort. 
The  simplest  of  all  ways  to  get  the  image  without  inversion  is  to 
arrange  the  slide  on  a  piece  of  white  paper  so  that  the  object  is  erect 
and  then  to  write  with  a  very  fine  pen  the  letters  a,  k,  on  the  cover- 
glass  of  the  specimen  to  be  drawn  (fig.  105).  Now  with  the  low 


FIG.  105.  SLIDE  OF  SERIAL  SECTIONS,  SHOWING  THE  DEVELOPMENT  OF  THE 
EYE  WITH  THE  LETTERS  a  k,  TO  Am  IN  GETTING  ERECT  IMAGES  LN  DRAWING 
WITH  PROJECTION  APPARATUS. 

(From  Optic  Projection). 

This  slide  is  also  to  show  how  to  mask  preparations  which  are  to  be  used  in  class 
demonstration  (Fig.  121). 

power  (16  to  60  mm.)  objective  project  the  image  of  the  specimen 
and  letters  upon  the  drawing  paper.  One  can  then  continue  to 
rearrange  the  slide  until  the  letters  are  erect;  the  specimen  will  then 
also  be  erect. 

§  278.  Images  to  be  traced  in  the  photographic  camera.  —  These 
images  are  wrong  side  up  and  the  rights  and  lefts  are  reversed.  This 
can  be  corrected  by  drawing  the  picture  on  the  tracing  paper  in  the 
inverted  position  and  then  inverting  the  tracing  after  it  is  finished; 
or  the  specimen  can  be  put  in  the  inverted  position,  then  the  image 
will  be  erect. 

Demonstrate  this  by  putting  the  metric  card  in  position  and  tracing 
some  of  the  larger  lettlrs  or  figures  on  the  tracing  paper.  Then 


174  AVOIDANCE  OF  INVERSION;    ERECT  IMAGES       [Cn.  VI 

turn  the  drawing  paper  around  180°  and  the  letters  or  figures  will 
appear  erect. 

Put  the  metric  card  wrong  edge  up  to  start  with;  then  the  letters 
or  figures  will  appear  right  side  up  on  the  tracing  paper. 

§  279.  The  use  of  a  negative  for  projection  and  tracing.  —  Put 
the  face  of  the  negative  that  reads  correctly  next  the  source  of  light 
and  wrong  edge  up :  then  it  will  appear  erect  in  every  way  on  the 
drawing  paper.  This  is  the  way  lantern  slides  are  put  in  the 
holder. 

§  280.  The  Wollaston  or  Abbe  camera  lucida.  —  With  these 
camera  lucidas  there  are  two  reflections  of  the  rays  (fig.  90-100), 
consequently  there  is  no  inversion  produced  by  the  camera,  but  the 
microscope  inverts  the  image  the  same  as  the  photographic  objective, 
and  erect  images  are  obtained  either  by  inverting  the  drawing  after 
it  is  made  or  by  putting  the  object  in  an  inverted  position  under 
the  microscope,  just  as  with  the  photographic  camera. 

Demonstrate  that  this  will  produce  erect  drawings  by  using  the 
letters  (fig.  105)  and  making  sketches  of  their  images  by  the  camera 
lucida,  having  the  letters  right  edge  up  on  the  stage  in  one  case  and 
wrong  edge  up  in  one. 

ERECT  IMAGES  WITH  THE  PROJECTION  MICROSCOPE 
§  281.  Erect  images  with  an  objective  only  or  with  an  objective 
and  amplifier. — There  are  two  cases:  (i)  When  opaque  drawing 
paper  is  used.  In  this  case  the  object  must  be  put  on  the  stage 
with  the  cover-glass  toward  the  light  and  the  slide  toward  the  ob- 
jective, and  it  must  be  wrong  edge  up.  Only  low  powers  (16  mm. 
and  lower  objectives)  should  be  used,  for  the  thick  slide  introduces 
aberrations  (fig.  51)  and  is  liable  to  be  too  thick  for  the  free  working 
distance  (fig.  31). 

(2)  When  a  translucent  drawing  paper  is  used  and  the  drawing 
is  made  on  the  back.  In  this  case  the  specimen  is  put  on  the  stage 
wrong  edge  up,  but  with  the  cover-glass  facing  the  objective.  All 
powers  can  be  used.  This  is  similar  to  the  conditions  described  for 
the  photographic  camera  where  the  tracing  paper  is  used  on  the  clear 
glass  (§  278). 


CH.  VI3        AVOIDANCE  OF  INVERSION;    ERECT  IMAGES 


175 


Test  the  correctness  of  the  directions  by  using  a  preparation  with 
the  letters  a,  k,  on  the  cover-glass  (§  277,  fig.  105). 

§  282.  Erect  images  with  an  objective  or  an  objective  and  an 
amplifier  and  a  prism  or  45°  mirror.  —  Place  the  specimen  on  the 
10  CENTIMETER  RULE 


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H3JLHJMIXN3D  Ol 
3CJLU5I  H3T3MITM3O  OI 


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4.  JO  CEMJLIIALEXEK  BIIPE 

FIG.  106.     i,  2,  3,  4,  ERECT  AND  INVERTED  IMAGES  OF  THE  METRIC  SCALE. 

(From  Optic  Projection). 
i.  Erect  image.     2.  Inverted  image.     3.  Mirror  image.     4.  Inverted  mirror  image, 

stage  wrong  edge  up  and  with  the  cover-glass  toward  the  objective. 

The  image  will  be  erect  on  the  opaque  drawing  paper.     Test  with 

the  lettered  specimen  (fig.  105). 

§  283.     Erect  images  with  an  objective  and  an  ocular.  - 

(i)   Opaque  drawing  paper.     Place   the   specimen   on   the   stage 


176  DRAWING  BY  THE  AID  OF  PHOTOGRAPHY         [Cn.  VI 

right  edge  up,  but  with  the  cover-glass  facing  the  light,  the  slide 
toward  the  objective. 

(2)  Translucent  drawing  paper.  If  the  drawing  can  be  made 
on  the  back  of  translucent  paper  the  specimen  is  placed  on  the  stage 
right  edge  up  and  with  the  cover-glass  facing  the  objective.  Test 
with  the  lettered  specimen  (fig.  105). 

§  284.  Erect  images  with  an  objective  and  ocular  and  a  45°  mirror 
or  prism.  —  Place  the  slide  on  the  stage  right  edge  up,  and  with 
the  cover-glass  facing  the  objective.  The  image  will  be  erect  on 
an  opaque  flrawing  surface.  Test  with  the  lettered  preparation 
(fig.  105).' 

'  DRAWINGS  BY  THE  AID  OF  THE  PHOTOGRAPHIC  CAMERA" 
AND  THE  MAGIC  LANTERN 

§  285.     Drawings  by  the   aid   of  a  photographic   camera.  —  The 

photographic  camera  (camera  obscura)  gives  help  for  getting  pictures 
of  objects  in  three  ways: 

(1)  By  producing  real  images  which  can  be  traced  (§  286). 

(2)  By  producing    negatives   which   can    be  projected  upon  the 
drawing  paper  and  traced,  or  the  drawing  can  be  done  directly  on 
the  print,  and  all  but  the  drawing  removed  from  the  print;   or  the 
drawing  can  be  made  on  the  back  of  the  print  (§  288-289). 

(3)  By  producing  large  prints  for  retouching  (§  290). 

§  286.  Real  images  by  the  camera.  —  For  drawing  with  a  photo- 
graphic camera  it  is  a  great  help  to  have  a  frame  with  a  piece  of 
clear  glass  to  use  instead  of  the  ordinary  ground-glass  focusing  screen. 
The  tracing  paper  is  stretched  over  the  glass.  The  object  is  arranged 
as  desired  and  placed  in  a  strong  light.  The  camera  is  then  arranged 
to  give  the  desired  view,  and  the  bellows  pulled  out  and  the  whole 
camera  moved  toward  or  away  from  the  object  until  the  desired  size 
is  obtained.  This  tracing  is  transferred  to  the  drawing  paper  in 
the  usual  manner  and  inked  in.  A  camera  like  that  shown  in  fig.  107 
answers  well;  also  a  copying  camera  (fig.  108). 

While  inking  in,  and  indeed  whenever  free-hand  and  optical  methods 
of  getting  drawings  are  combined  the  object  should  be  available  for 
constant  observation  so  that  accuracy  may  be  obtained. 


CH.  VI]          DRAWING  BY  THE  AID  OF  PHOTOGRAPHY 


177 


§  287.     Negatives  by  the   camera.  —  The  object   is  arranged  as 
desired  and  placed  in  a  good  light.     A  photographic  camera  is  then 


FIG.  107.    VERTICAL  PHOTOGRAPHIC  CAMERA  ON  A  Low  TABLE. 

T    Table  about  50  cm.  high  and  50  cm.  by  70  cm.  on  the  top. 

cl  d     Drawer  with  combination  lock. 

Base    The  heavy  base  of  the  vertical  camera  support. 

/>     Pillar  in  which  the  graduated  rod  (vgr)  rotates. 

ss     Set  screw  to  fix  the  graduated  rod  in  any  position. 

c  s,  c  s  Set  screws  to  enable  the  operator  to  set  the  camera  bellows  at  any 
desired  extension. 

mr  Magnification  rod  with  its  set  screw  rs.  When  any  desired  magnification 
is  arranged,  the  rod  set  screw  is  tightened;  then  by  loosening  the  camera  set  screws 
(cs)  the  bellows  can  be  moved  up  and  down  on  the  graduated  rod  to  get  the  focus. 

Ps  Focusing  stand;  this  is  a  microscope  stand  with  coarse  and  fine  adjust- 
ment (cf)  and  two  stages  ($/,  si)  for  supporting  the  object  or  the  dish  containing 
it  (sp  c) 

Ob     Photographic  objective  in  the  lower  end  of  the  camera. 

VC    Vertical  camera  bellows. 

/g     Focusing  glass. 

used  and  a  negative  on  glass  made  in  the  usual  manner.     If  the 
negative  is  to  be  used  for  prints  on  which  to  trace  and  draw  with 


i78 


DRAWING   BY  THE  AID  OF  PHOTOGRAPHY          [Cn.  VI 


ink  or  pencil,  the  negative  is  made  the  size  of  the  desired  finished 
picture.  On  the  other  hand  if  the  negative  is  to  be  used  for  projec- 
tion, it  should  be  of  about  the  size  of  a  lantern  slide  (§  290). 

§  288.  Drawings  upon  blue  prints.  —  This  is  especially  available 
for  objects  with  definite  outlines  and  clear  details  like  the  wing  veins 
of  insects  (Comstock)  or  apparatus,  furniture,  etc. 

A  negative  of  the  object  is  made  of  the  desired  size  and  a  blue 
print  made.  Then  with  waterproof  India  ink  all  the  lines  are  gone 


.0 

O 


Base 


FIG.  108.     CAMERA  OBSCURA  FOR  DRAWING,  AND  LANTERN  SLIDE  MAKING. 
(From  Optic  Projection). 

over,  and  all  the  points  indicated  which  are  to  be  shown  in  the  finished 
cut. 

Bleach  out  the  blue  by  soaking  the  print  in  a  solution  of  10% 
neutral  oxalate  of  potash.  Wash  in  water  and  dry  on  gauze.  Only 
the  ink  lines  will  show  in  the  finished  print.  This  line  drawing  can 
then  be  lettered  in  any  desired  way,  and  the  engraver  can  make  a 
line  cut  for  the  printing  press. 

Ordinarily  it  is  best  to  make  the  picture  two  or  three  times  the 
size  of  the  final  engraving.  Defects  are  minimized  in  the  reduction. 
Always  have  the  object  in  view  in  finishing  the  drawing. 

§  289.  Drawings  on  the  back  of  photographic  prints.  —  Instead 
of  making  a  blue  print,  a  photographic  print  can  be  made  of  the 
negative  of  the  object  to  be  drawn  in  lines.  Use  double  thick  devel- 
oping paper  (Cyco,  Velox,  etc.). 

For  this  the  best  method  is  to  make  a  small  negative  of  about  the 
size  of  a  lantern  slide,  using  a  rather  long  focus  objective  so  that  all 


CH.  VI]          DRAWING  BY  THE  AID  OF  PHOTOGRAPHY 


179 


parts  will  be  in  focus  and  in  proper  perspective.     Then  with  the 
projection  apparatus  using  a  photographic  objective,  print  an  enlarged 


FIG.  109.    PROJECTION  MICROSCOPE,  TABLE,  AND  ADJUSTABLE  DRAWING  SHELF. 
(Modified  from  Optic  Projection). 

DB  Drawing  board  with  a  25  X  30  cm.  glass  plate  in  the  middle  for  tracing 
on  the  back  of  photographs.  It  is  placed  on  the  brackets  to  form  the  adjustable 
shelf  (ADS). 

Is    Leveling  screws  in  the  bottom  of  the  table  legs. 

Rheostat    The  balance  for  regulating  the  electric  current  of  the  arc  lamp. 

c  c,  ks     Electric  cable  and  knife  switch. 

Table  The  projection  table  with  drawer  (d).  This  table  is  100  cm.  high,  and 
the  top  125  cm.  long  and  50  cm.  wide.  It  is  stained  by  aniline  black. 

ADS    Adjustable  shelf  with  a  drawing  board  having  a  glass  center  25  X  30  cm. 

bt     Bolts  with  thumb  nuts  holding  the  shelf  at  any  desired  height  on  the  legs. 

N  R  Mazda  lamp  and  reflector  to  throw  the  light  up  through  the  picture  which 
is  being  traced. 

c     Cable  with  separable  cap  to  attach  to  the  lighting  system. 

Arc  Lamp  The  right-angled  carbon  arc  lamp  for  supplying  light  to  the  pro- 
jection microscope. 

Condenser    The  three  lens  condenser  and  water  bath  (fig.  no). 

Microscope    The  compound  microscope  with  substage  condenser  and  ocular. 

m  45°  mirror  or  prism  for  reflecting  the  light  directly  downward  upon  the 
drawing  shelf. 

Axis,  Axis    The  principal  optic  axis. 

picture  as  follows:  Work  in  a  dark  room  or  at  night.  Place  the 
negative  near  the  condenser  as  for  lantern  slide  projection  (fig.  no). 
Either  the  projection  apparatus  must  be  movable  of  a  movable 


i8o  DRAWING  BY  THE  AID  OF  PHOTOGRAPHY          [Cn.  VI 

screen  must  be  used  to  get  the  desired  size,  which  should  be  two, 
three,  or  four  times  the  size  of  the  final  picture.  Use  a  large  printing 
frame,  one  25  X  30  or  28  X  35  cm.  (10  X  12  or  n  x  14  in.).  Place 
the  printing  frame,  in  which  are  a  clear  glass  and  a  white  sheet  of 
paper,  against  the  wall  or  movable  screen,  and  by  moving  the  screen 
or  the  apparatus  get  the  picture  the  desired  size.  Now  focus  very 
sharply.  The  diaphragm  of  the  objective  must  be  wride  open.  Turn 
off  the  light  from  the  arc  lamp  and  place  in  the  printing  frame  a 
sheet  of  the  photographic  paper.  Place  a  piece  of  ruby  glass  over  the 
end  of  the  projection  objective,  turn  on  the  light,  and  then  arrange 
the  printing  frame  so  that  the  picture  is  in  the  desired  position.  Re- 
move the  ruby  glass  and  give  an  exposure  of  2  to  5  seconds  for  the 
arc  light 'or  considerably  more  for  a  stereopticon  mazda  lamp.  Re- 
place the  ruby  glass  over  the  objective,  turn  off  the  light,  and  develop 
the  picture  as  usual.  A  good  plan  to  follow  is  to  put  a  small  piece 
of  paper  in  the  printing  frame  and  test  the  exposure  before  putting 
the  large  sheet  in  the  frame.  The  paper  is  too  expensive  to  use  the 
large  sheets  for  trial  exposures. 

When  the  prints  are  developed,  fixed  and  dried,  the  drawing  in 
lines  is  made  as  follows:  Use  a  drawing  board  with  a  piece  of  plane 
glass  in  the  middle  (fig.  109)  as  the  drawing  shelf,  and  have  under 
it  an  incandescent  lamp  and  metal  reflector  (fig.  109).  Fix  the  print 
face  down  on  the  drawing  board  and  glass.  The  light  from  the 
lamp  shines  through  the  paper  and  one  can  see  the  picture  almost 
as  clearly  as  by  looking  at  the  face  of  the  print.  Now  with  the 
T-square,  etc.,  put  in  the  lines  desired.  For  a  beginner  it  is  best  to 
do  this  with  pencil.  Then  the  pencil  sketch  can  be  inked  in  at  any 
time  in  the  usual  manner.  While  penciling  in  the  lines  the  light 
should  be  turned  off  occasionally  so  that  the  pencil  marks  can  be 
seen  clearly;  then  one  can  see  whether  any  essential  parts  have  been 
omitted.  The  photographic  paper  is  of  excellent  quality  and  takes 
the  right  line  pen  almost  as  well  as  the  best  drawing  paper.  The 
thick  paper  is  used  so  that  the  photographic  print  will  not  show 
through,  and  because  the  thick  paper  holds  its  form  better  than  the 
thinner  paper.  The  thinner  paper  will  also  answer. 

One  can  use  this  method  with  blue  prints  also.     It  has  the  ad- 


CH.  VI]          DRAWING  BY  THE  AID  OF  PHOTOGRAPHY  181 

vantage  that  the  lines  are  perfectly  distinct  in  every  stage  of  the 
work.  If  drawn  on  the  face  of  the  print  the  blue  obscures  the  pencil 
lines  more  or  less  while  the  drawing  is  being  made. 

For  many  objects  it  makes  no  great  difference  whether  the  picture 
is  reversed  or  not,  but  in  some  cases  there  should  be  no  reversal. 
One  can  easily  make  the  picture  so  that  the  final  picture  will  be 
erect  as  follows:  Print  the  negative  so  that  the  photograph  will  be 
reversed;  then  when  the  line  drawing  is  made  on  the  back  of  the 
print  the  line  drawing  will  be  erect.  Of  course  if  the  lines  are  made 
on  the  face  of  the  print  as  with  the  blue  print  (§  288)  the  print  must 
be  erect.  To  get  erect  prints  turn  the  film  side  of  the  negative  toward 
the  sensitive  paper  as  with  contact  printing.  For  reversed  prints 
turn  the  glass  side  of  the  negative  toward  the  paper. 

This  method  of  drawing  is  applicable  for  all  sorts  of  objects,  the 
photographic  print  serving  to  give  all  the  outlines  and  proportions. 
No  measurements  need  be  made.  Then  by  drawing  the  outlines 
on  the  back  of  the  print,  one  can  do  all  shading  as  if  no  picture  were 
on  the  opposite  side.  It  is  of  course  not  necessary  for  highly  trained 
artists,  but  is  of  the  greatest  assistance  for  amateurs;  and  most 
biologists  are  amateurs. 

In  finishing  the  drawing,  the  object  should  be  in  view  to  make 
certain  that  the  drawing  is  accurate. 

§  289a.  Diaphragming  the  objective  and  the  use  of  a  concentrated  filament 
lamp.  —  In  the  above  directions  a  first-class  photographic  objective  was  assumed. 
If  now  one  has  not  a  first  class  objective  or  for  any  reason  it  is  desirable  to  close 
the  diaphragm  more  or  less,  the  unobstructed  cone  of  light  cannot  be  used,  but 
there  must  be  a  diffuser  like  ground-glass  or  milky  glass  put  between  the  source 
of  light  and  the  negative  to  be  projected.  With  such  a  diffuser  one  can  close  the 
diaphragm  as  desired.  Of  course  the  addition  of  the  diffuser  and  the  closing  of 
the  diaphragm  will  necessitate  a  longer  exposure. 

If  instead  of  an  arc  lamp  a  concentrated  filament  stereopticon  lamp  is  used  one 
must  also  employ  a  diffuser  or  the  shadows  between  the  filaments  of  the  lamp  will 
give  rise  to  inequalities  in  the  print.  The  diffuser  can  be  put  between  the  con- 
denser lenses  or  between  the  lamp  and  the  condenser.  It  must  be  far  enough  from 
the  negative  so  that  the  grain  of  the  ground-glass  will  not  show  in  the  print  (§  362). 

§  290.  Retouching  photographs  for  halftone  reproduction.  —  For 
pictures  of  animals,  organs,  and  dissections  to  be  reproduced  by  the 
halftone  process,  very  successful  drawings  can  be  made  as  follows: 
Arrange  the  object  as  it  is  to  appear  in  the  finished  drawing;  light  it 


182  DRAWING  BY  THE  AID  OF   PHOTOGRAPHY         [Cn.  VI 

to  bring  out  clearly  the  features  desired;  then  use  a  long  focus  photo- 
graphic objective  and  get  a  small,  sharp  picture.  The  negative 
should  be  about  the  size  of  a  lantern  slide,  and  it  should  be  a  good 
printing  negative.  Make  a  large  print  on  thick  developing  paper 
exactly  as  described  in  the  previous  section  (§  289).  This  print 
should  not  be  dark,  but  two  or  three  shades  lighter  than  the  usual 
print  to  give  opportunity  for  the  added  shading.  The  picture  should 
be  erect. 

When  the  print  is  dry,  put  it  on  a  drawing  board  and  with  a  carbon 
drawing  crayon,  pen,  India  ink,  and  an  air  brush,  if  it  is  available, 
the  picture  can  be  made  almost  perfect  with  a  minimum  of  labor. 

In  case  the  negative  shows  parts  not  needed  or  if  the  background 
is  not  as  desired,  the  superfluous  parts  can  be  eliminated  and  the 
background  made  perfectly  white  by  painting  on  the  glass  surface 
of  the  negative  Gihon's  or  other  opaquing  medium.  In  the  print 
there  will  be  pure  white  where  the  opaque  is  painted  on  the  glass. 
Use  a  fine  brush  and  put  on  a  layer  which  does  not  allow  any  light 
to  pass.  The  opaque  is  put  on  the  glass  surface  so  that  it  can  be 
easily  removed  if  desired.  In  case  some  parts  are  not  light  enough 
or  white  points  are  to  be  added,  use  some  of  the  white  recommended 
by  the  photo-engravers  (Blanc  d'Argent  etc.) . 

As  in  all  drawing,  the  actual  object  should  be  before  the  artist 
when  retouching  the  photograph,  so  that  accuracy  may  be  secured. 

§  291.  Tracing  pictures  natural  size  on  drawing  paper.  —  It 
frequently  happens  in  preparing  the  drawings  for  a  book  or  for  a 
scientific  paper  that  figures  from  another  book  or  from  a  scientific 
paper  are  needed.  If  there  is  to  be  no  modification  in  the  figure  the 
simplest  method  is  to  borrow  an  electrotype.  If  this  cannot  be 
done  and  the  picture  is  not  available  to  put  in  the  hands  of  the  photo- 
engraver  for  a  new  cut,  or  if  one  wants  to  make  minor  changes,  it  is 
very  easy  to  get  a  tracing  on  any  good  drawing  paper  as  follows: 
Put  the  picture  on  the  glass  of  the  drawing  shelf  (fig.  IOQ)  and  place 
over  it  some  good  drawing  paper  like  Whatman's  hot-pressed  drawing 
paper  or  Reynolds  bristol  board.  Turn  on  the  light,  and  even  through 
the  thick  drawing  paper  the  outlines  of  the  picture  are  so  clear  that 
the  tracing  can  be  made  with  ease.  After  the  outlines  have  been 


CH.  VI]          DRAWING  BY  THE  AID  OF  PHOTOGRAPHY 


183 


traced,  the  finishing  can  be  done  on  a  drawing  board,  having  the 
original  picture  for  reference. 

§  292.  Drawings  by  a  projection  or  a  photographic  objective.  — 
For  light  use  an  arc  lamp  or  a  stereopticon  mazda  lamp;  use  a  nega- 
tive which  is  not  too  dense  or  a  lantern  slide.  It  is  placed  in  the 
lantern-slide  holder  and  by  means  of  an  ordinary  projection  objec- 
tive, or  better  by  a  photographic  objective,  the  image  is  projected 
upon  the  drawing  paper  (fig.  no).  For  the  proper  size  either  the 


Condenser 


FIG.  no.    MAGIC  LANTERN  WITH  PROJECTED  IMAGE. 
(From  Optic  Projection). 

A  small  arc  lamp  connected  with  the  house  lighting  system  is  used  for  light  in 
this  case. 

W,  So,  S  —  p  Electric  wires,  lamp  socket  with  key  switch  (s)  and  a  separable 
attachment  plug. 

R     Rheostat. 

Condenser,  W    A  three  lens  condenser  with  a  water  cell  to  absorb  radiant  heat. 

LS    Lantern  slide. 

Axis,  Objective  The  principal  optic  axis  of  the  condenser  and  of  the  objective 
in  one  line.  The  cone  of  light  crosses  within  the  objective  at  (c). 

Screen  Image    The  real  image  projected  upon  the  screen. 

projection  apparatus  or  the  drawing  surface  must  be  movable.  For 
most  artists  it  is  better  to  make  the  drawing  two  or  three  times  the 
size  which  it  is  to  have  after  engraving.  The  reduction  minimizes 
the  little  irregularities  which  are  almost  sure  to  be  present. 

When  the  size  is  correct,  and  the  image  sharply  focused,  one  can 
trace  directly  on  the  drawing  paper  with  a  pencil  all  the  lines  and 
details  which  it  is  desired  to  represent.  Then  the  drawing  can  be 
inked  in  at  leisure,  remembering  always  to  have  the  object  for  con- 
stant reference  and  thus  insure  accuracy. 

In  making  the  negatives  for  projection  it  is  very  desirable  that 


1 84  DRAWING  WITH  A  PROJECTION  MICROSCOPE         [Cn.  VI 

the  photographic  objective  should  be  of  rather  long  focus  and  thus 
make  it  possible  to  have  the  camera  at  a  considerable  distance  from 
the  object;  then  there  will  be  avoided  the  exaggerated  perspective 
which  comes  from  using  a  short  focus  objective. 

All  the  different  objects  or  parts  of  a  large  object  at  different  levels 
will  be  in  focus  with  the  long  focus  objective  at  a  considerable  distance. 
In  projection  it  is  very  easy  to  make  the  picture  as  large  as  desired 
provided  the  projection  apparatus  or  the  drawing  surface  is  movable. 
The  projection  method  has  the  advantage  of  being  applicable  to  all 
forms  of  objects,  gross  and  microscopic.  The  only  precaution  is  to 
make  the  negative  rather  thin,  not  dense;  then  the  details  come  out 
clearly  in  the  projected  image. 


PROJECTION  MICROSCOPE  FOR  DRAWING 

§  293.  This  is  the  most  satisfactory  method  of  drawing  small 
objects.  With  it  one  can  draw  large  diagrams  or  small  figures 
directly  from  the  objects;  and  if  the  apparatus  is  properly  con- 
structed one  may  make  diagrams  from  objects  60  to  70  mm.  in  di- 
ameter down  to  those  of  half  a  millimeter  or  less.  This  method  was 
much  in  vogue  and  highly  commended  by  the  older  microscopists 
who  used  the  solar  microscope  (Baker,  Adams,  and  Goring).  Since 
the  general  introduction  of  electric  lighting,  drawing  with  the  pro- 
jection microscope  has  become  once  more  common  and  is  the  most 
satisfactory  method  known,  especially  for  the  numerous  drawings 
necessary  for  the  preparation  of  models  in  wax  or  blotting  paper. 

§  294.  Drawings  with  low  powers.  —  For  objectives  of  30  to  100 
mm.  focus  the  best  method  is  to  use  a  projection  outfit  with  a  three 
lens  condenser  as  shown  in  fig.  in.  The  whole  should  be  on  an 
optical  bench  so  that  each  element  and  all  together  can  be  moved  at 
will  (fig.  131). 

For  a  radiant  a  large  or  a  small  arc  lamp  is  best  (fig.  49,  111-112), 
but  a  250  or  400  watt  concentrated  filament,  stereopticon  mazda 
lamp  filled  with  nitrogen  also  works  fairly  well.  It  has  the  ad- 
vantage that  it  can  be  attached  to  any  lighting  circuit,  and  when 
once  centered  and  properly  arranged  requires  no  attention  except  to 


CH.  VI]       DRAWING  WITH  A  PROJECTION  MICROSCOPE 


185 


turn  the  switch  on  and  off.     A  dark  room  is  desirable,  but  one  can 
draw  in  any  room  at  night. 

Arrange  the  object,  the  lamp,  and  the  condenser  so  that  the  object 
is  fully  lighted;  then  focus  the  objective  and  place  the  drawing  surface 
and  objective  at  a  distance  apart  to  give  the  desired  size  of  drawing. 
Focus  sharply  and  trace  with  a  pencil  the  outlines  and  details  which 
it  is  desired  to  show.  Finally,  with  the  object  where  it  can  be  ex- 


FIG.  in.    PROJECTION  MICROSCOPE. 
(From  Optic  Projection). 

+  w  The  positive  wire  going  to  the  upper  carbon  (He) ,  and  —  u>,  wire  to  the 
lower  or  vertical  carbon  (Vc)  of  the  large  arc  lamp  with  direct  current. 

Axis,  Axis,  Axis  The  principal  optic  axis  from  the  source  of  light  (L)  through 
the  condenser,  the  microscope  and  to  the  screen. 

W     Water  cell  to  absorb  radiant  heat. 

Stage  The  separate  stage  of  the  microscope  with  its  water  cell  for  cooling 
the  specimen  by  induction. 

Microscope  In  this  case  the  microscope  has  an  objective  only;  compare  fig. 
109,  where  an  ocular  is  present  also. 

Each  element,  lamp,  condenser,  stagehand  microscope  is  on  a  separate  movable 
block  (block  i,  2,  3,  4)  which  slides  independently  along  the  optic  bench  or  base 
board  (fig.  131). 

amined  at  any  time,  ink  in  the  lines  and  details  (for  erect  images  see 
§  282). 

§  295.  Use  of  a  45°  mirror  or  a  prism.  —  While  one  can  draw  on 
a  vertical  surface  it  is  far  easier  to  draw  on  a  horizontal  surface. 
This  is  available  for  all  powers  by  using  a  plane  mirror  at  45°  or  a 
drawing  prism.  The  mirror  may  be  at  a  distance  from  the  objec- 
tive, when  it  must  be  large  (fig.  112),  or  it  may  be  close  to  the  objec- 
tive, when  it  may  be  small  (fig.  109,  114).  The  drawing  surface 
must  be  movable  to  vary  the  size  of  the  drawing  and  the  magnifica- 
tion. Figures  109,  111-112  show  the  two  principal  methods  of 


1 86 


DRAWING  WITH  A  PROJECTION  MICROSCOPE       [Cn.  VI 


varying  the  distance  between  the  objective  and  the  drawing  surface, 
and  consequently  the  scale  of  the  drawing  (for  erect  images  see  §277- 
284). 

§  296.     Drawing  with  objectives  of  25  to  10  mm.  focus.  —  For  this 
the  best  way  is  to  use  a  three  lens  condenser,  as  shown  in  fig.  109,  in, 

and  for  a  microscope  use 


either  the  special  water- 
cell  stage  or  the  ordinary 
microscope  with  large 
tube.  For  radiant  use  a 
small  or  a  large  arc  lamp. 
Remove  the  substage  con- 
denser or  turn  it  aside  and 
arrange  on  the  optical 
bench  so  that  the  image 
of  the  light  source  from 
the  large  condenser  falls 
directly  on  the  specimen. 
Focus  and  arrange  the 
drawing  surface  to  give 
the  right  size  and  mag- 
nification, then  trace  the 
outlines  and  the  details. 
Later,  ink  in,  using  the 
specimen  to  check  up  with 
(for  erect  images  see  §  277- 
284). 


FIG.  112.    PROJECTION  MICROSCOPE  WITH  MOV- 
ABLE DRAWING  TABLE  AND  45°  MIRROR. 

(From  Optic  Projection). 

The  projection  table  has  the  dimensions  given 
in  fig.  109. 

The  arc  lamp  is  automatic  and  the  rheostat 
for  current  varying  from  10  to  20  amperes. 

The  condenser  is  of  the  three  lens  water  cell 
type,  and  the  microscope  with  separate  stage; 
the  microscope  has  an  amplifier  in  place. 

The  drawing  table  (Dr.  Table)  is  of  a  conven- 
ient height  for  sitting  beside.  It  is  76  cm.  high 
and  the  top  100  cm.  long  and  75  cm.  wide. 

The  45°  plate  glass  mirror  is  large  (75  cm.  long 
and  60  cm.  wide). 


After  one  has  had  sufficient  practice,  the  drawing  can  be  partly 
or  wholly  completed  under  the  projection  apparatus.  For  this  one 
must  light  the  drawing  surface  enough  either  by  means  of  a  portable 
lamp  or  by  some  means  of  letting  in  daylight.  At  the  same  time 
there  must  be  a  screen  to  cut  off  the  image  where  one  is  doing  the 
finishing.  By  removing  the  screen  the  image  appears  at  any  time 
and  serves  to  check  the  work. 

§  297.  Drawing  with  high  powers,  8  to  2  mm.  focus.  —  For  this 
high  power  drawing  one  should  use  an  ocular  as  well  as  an  objective, 


CH.  VI] 


DRAWINGS  FOR  PUBLICATION 


187 


and  a  substage  condenser  in  addition  to  the  condenser  of  the  lantern 
or  small  lamp  (fig.  49,  114),  or  light  of  sufficient  aperture  will  not 
be  supplied  to  the  microscope.  In  using  the  highest  powers  it  is 
also  well  to  connect  the  substage  condenser  to  the  slide  by  homo- 
geneous liquid,  as  described  in  §  113.  The  large  or  small  arc  light 
is  the  only  really  satis- 
factory radiant  (for  erect 
images  see  §  283). 

If  one  has  a  drawing 
room,  a  large  or  small  arc 
lamp,  and  direct  current, 
the  arrangements  shown 
in  fig.  109  are  best,  but 
if  direct  current  is  not 
available,  excellent  results 
can  be  obtained  by  using 
the  small  arc  lamp  on  the 
alternating  current  house 
electric  lighting  system 
and  the  microscope,  as 
shown  in  fig.  113-114. 


FIG.  113.    DRAWING  MICROSCOPE  WITH  SMALL 
ARC  LAMP  ON  THE  HOUSE  LIGHTING  SYSTEM. 

(From  Optic  Projection). 


S,  Sp    The  lamp  socket  and  separable  attach- 
ment plug. 

Rh    The  rheostat  not  allowing  over  5  amperes 
of  current  to  flow. 

The    light    supplied    to        Lamp    The  small  arc  lamp  (fig.  49),  at  right 
the     substage     condenser    angles  to  the  microscope. 

should  be  approximately 
parallel.  This  is  attained 
with  the  small  lamp  by 


Microscope    The  microscope  on  a  block  (B). 

mr,  mr  The  mirror  of  the  microscope  and  the 
mirror  over  the  ocular  to  reflect  the  light  directly 
downward. 

Image    The  picture  of  the  microscopic  object 


putting   the    arc   at    the 
focus  of  the  condenser  (fig. 


reflected  down  upon  the  drawing  paper. 
Sh    Opaque  shield  to  screen  the  light  from  the 


drawing  surface. 
49) .     With  the  large  lamp 

one  should  use  a  long  focus  lens  for  the  condenser,  as  shown  in  fig.  115. 

In  all  cases  the  substage  condenser  should  be  shifted  up  and  down 

slightly  until  the  best  effect  is  produced.     The  substage  condenser 

should  of  course  be  carefully  centered  before  commencing  to  draw 

(§  104). 

§  298.    Drawings  for  publication.  —  The  inexpensive  photographic 
processes  of  making  cuts  for  the  printing  press  bring  within  the  reach 


i88 


DRAWINGS  FOR  PUBLICATION 


[CH.  VI 


of  every  writer  the  possibility  of  appealing  to  the  eye  by  means  of 
pictures  and  diagrams  illustrating  the  facts  which  are  presented  in 
the  text.  Artistic  ability  is  of  course  indispensable  for  a  perfect 
representation,  but  any  one  willing  to  give  the  time  and  the  pains 


Substage 
Condenser 


FIG.  114.    THE  MICROSCOPE  ARRANGED  FOR  DRAWING  ON  A  HORIZONTAL 

SURFACE. 

(From  Optic  Projection). 

The  microscope  is  of  the  handle  type  (H)  with  the  fine  adjustment  (/  a)  on  the 
side  below  the  coarse  adjustment  (c  a). 

The  ocular  is  of  the  Huygenian  form  with  the  real  image  at  (r  i). 

Prism,  the  right-angled  prism  beyond  the  ocular  to  reflect  the  light  directly 
downward. 

can  make  simple  drawings,  especially  if  one  or  more  of  the  helps 
above  described  are  available. 

The  various  helps  for  making  drawings  described  in  this  chapter 
will  be  found  useful  to  the  born  artist  as  well  as  to  the  person  who 
has  not  great  artistic  ability,  for  by  means  of  the  optical  and  mechani- 
cal helps  the  outlines  and  proportions  can  be  secured  with  fidelity  by 
any  one.  Then  the  born  artist  can  use  the  time  saved  for  making 
the  pictures  more  artistic,  and  the  plodder  can  feel  confident  that 
his  efforts  are  correct  even  if  not  pretty. 

Young  authors  are  urged  to  get  the  Style  Brief  furnished  by  the 
Wistar  Institute  of  Philadelphia.  This  is  a  guide  for  the  preparation 


CH.  VI] 


DRAWINGS  FOR  PUBLICATION 


189 


of  manuscript  and  drawings  for  publication  in  the  scientific  journals 
published  by  the  Institute.  The  hints  to  contributors  given  on 
the  second  page  of  the  cover  in  all  the  journals  give  in  a  nutshell  the 


FIG.  115.    DIAGRAMS  TO  SHOW  THE  POSITION  OF  THE  SUBSTAGE  CONDENSER 
WHEN  NO  PARALLELIZING  LENS  is  USED. 

(From  Optic  Projection.) 

A  The  substage  condenser  is  within  the  focus  (/)  at  a  point  where  the  long 
light  cone  is  of  about  the  same  diameter  as  the  substage  condenser. 

B  The  substage  condenser  is  beyond  the  focus  (/)  of  the  long  focus  main  con- 
denser at  a  point  where  the  diverging  cone  is  of  about  the  same  diameter  as  the 
substage  condenser.  This  is  the  better  position  for  the  substage  condenser  of  the 
ordinary  microscope. 

Arc  Supply    The  right-angled  carbons  of  the  arc  lamp. 

L\  Lz    The  first  and  the  second  elements  of  the  main  condenser. 

Water  Cell.     This  is  to  remove  the  radiant  heat. 

Axis    The  principal  axis  on  which  all  the  parts  are  centered. 

/  The  principal  focus  of  the  second  element  of  the  main  condenser.  In  both 
cases  the  focus  is  long. 

Substage  Condenser  This  is  the  first  or  lowest  element  of  the  substage  con- 
denser. It  is  of  the  achromatic  type. 


main  points.  These  journals  are:  The  American  Journal  of  Anat- 
omy; The  Anatomical  Record;  The  Journal  of  Morphology;  The 
Journal  of  Comparative  Neurology,  and  the  Journal  of  Experimental 
Zoology. 


I  go  DRAWINGS  FOR  PUBLICATION  [Cn.  VI 

A  great  many  good  hints  can  be  found  by  studying  the  illustrations 
in  well-printed  books  and  in  scientific  journals,  especially  those 
dealing  with  the  subject  in  which  one  is  interested. 

§  299.  Outlining  and  inking  in.  —  In  making  drawings  two  steps 
are  necessary  for  all  but  the  most  expert:  (i)  getting  the  outlines 
and  main  details  in  pencil,  and  (2)  inking  the  outlines  and  details. 

For  the  outlining  one  or  more  of  the  helps  described  above  will  be 
found  almost  indispensable.  For  the  inking  in  the  draughtsman 
should  have  the  actual  object  for  constant  reference  so  that  the 
representation  may  be  accurate.  The  final  work  in  inking  is  almost 
always  done  with  a  right  line  pen,  free-hand,  and  of  course  in  a  good 
light  and  convenient  position.  If  one  would  make  colored  pictures 
it  is  best  to  get  the  guidance  and  criticism  of  an  expert. 

One  should  keep  in  mind  the  way  to  make  the  picture  erect  when 
using  any  of  the  helps  described  (§  277-284). 

§  300.  Size  of  drawings.  —  For  most  draughtsmen  it  is  wise  to 
make  the  drawings  two  or  three  times  the  size  of  the  final  cut  for 
publication.  It  is  easier  to  make  the  details  clear,  and  then  little 
defects  are  minimized  by  the  reduction.  The  photo-engraver  can 
make  the  cut  any  desired  reduction,  but  one  should  remember  that 
the  lines  should  be  heavy  enough  for  the  reduction  desired,  otherwise 
the  finest  details  are  liable  to  be  lost. 

§  301.  Reduction.  —  There  is  some  confusion  as  to  the  meaning 
of  reduction  in  the  minds  of  authors.  For  the  engraver  this  term 
has  a  perfectly  definite  significance.  It  is  linear  measure,  and  never 
area  or  solid  measure,  that  he  considers.  For  example,  if  the  en- 
graver is  directed  to  make  the  cut  half  the  size  of  the  drawing  he 
will  make  every  line  half  the  length  of  the  corresponding  line  in  the 
drawing.  The  area  will  then  be  one-fourth  that  of  the  drawing. 
If  the  cut  is  to  be  reduced  to  one-fourth  the  drawing,  each  line  will 
be  only  one-fourth  the  length  of  the  original,  and  the  area  will  be 
one-sixteenth  that  of  the  drawing  (fig.  116). 

§  302.  Lettering  drawings.  —  After  the  drawings  are  finished  the 
details  must  be  indicated  in  some  way.  This  may  be  by  having  the 
full  name  of  the  part,  an  easily  intelligible  abbreviation,  or  a  letter 
or  a  numeral  upon  or  near  it  (fig.  106). 


CH.  VI]  DRAWINGS  FOR  PUBLICATION  191 

The  lettering  should  be  done  with  discrimination  in  two  ways: 

(1)  The  letters,  words,  etc.,  should  be  artistically  arranged  and 
then  put  on  straight.     For  this  one  may  need  to  use  a  T-square  and 
straight  edge.     Most  persons  cannot  letter  neatly  enough  to  letter 
with  a  pen.     Printed  words  and  letters  can  be  pasted  upon  the  draw- 
ing.    In  the  final  cut  the  appearance  is  as  if  words,  letters,  or  numerals 
were  printed  on  the  picture  (fig.  25). 

If  the  letters,  abbreviations,  etc.,  are  not  upon  the  parts  they  are 
meant  to  indicate,  then  "leaders,"  that  is,  full  or  broken  lines  should 
be  drawn  from  the  part  to  its  designating  letter,  numeral,  abbrevia- 
tion, or  word  (fig.  20,  25). 

(2)  The  size  of  type  to  be  used  should  correspond  to  the  size  of 
the  picture  and  the  amount  of  reduction.     The  letters  should  not  be 
the  most  prominent  thing  about  a  picture,  neither  should  they  be  so 
small  that  one  needs  a  microscope  to  read  them.     By  consulting 
fig.  116  one  can  get  a  clear  notion  of  the  appearance  of  various  sizes 
of  letters  when  reduced.     If  one  has  a  camera  (fig.  107),  it  is  a  good 
plan  to  put  letters  of  different  sizes  upon  the  drawing  and  then, 
having  the  bellows  set  to  give  the  reduction  desired,  look  at  the  image 
of  the  drawing  and  lettering  and  see  how  they  will  look  in  the  final 
picture. 

For  photo-engraving,  Gothic  type  gives  the  best  results  (fig.  116). 

§303.  Fastening  the  letters  to  the  drawing. —The  letters,  etc., 
should  be  printed  on  thin,  smooth,  very  white  paper,  and  they  should 
be  black,  not  gray.  Tissue  paper  is  often  used,  but  that  is  not  so 
easy  to  handle  as  a  paper  about  like  the  so-called  "Bible  paper." 

The  words,  letters,  and  numerals  for  a  drawing  are  cut  out  and 
arranged  on  the  drawing  to  get  the  best  effect.  Then  using  a  T- 
square  and  straight  edge  each  letter  or  word  is  stuck  to  the  drawing 
in  the  proper  position  as  follows:  Some  fresh  starch  paste  is  made 
by  placing  in  a  small  tin  or  aluminum  dish  5  grams  of  laundry  starch 
and  adding  50  cc.  of  cold  water.  Stir  with  a  spoon  and  then  heat 
gradually  with  constant  stirring  on  a  stove  or  over  a  gas  flame  until 
the  paste  is  formed.  Mucilage  and  paste  which  has  been  made  for 
some  time  are  not  good  for  pasting  the  letters.  Mucilage  turns  the 
paper  yellow  and  the  old  paste  is  lumpy. 


192  DRAWINGS  FOR  PUBLICATION  [Cn.  VI 

Use  a  fine  brush  to  put  the  paste  on  the  letters,  and  then  use  fine 
forceps  (fig.  70)  to  pick  up  the  letters  and  transfer  them  to  the  proper 
position.  Press  down  with  the  finger  covered  with  tissue  paper  or 
very  fine  cloth  or  with  fine  blotting  paper.  Press  directly  downward 
or  the  letter  is  liable  to  be  displaced  or  distorted  by  a  lateral  thrust. 


24  Point  Type   A  a 
123456789 10 

18  Point  Type  ARS  2  34 

12  Point  Type    ABCabc    1234 
10  Point  Type     ABC     abc     12345 

8  Point  Type    ABCD      abed      12345 

6  Point  Type       A    B    C    D    a    b    c    d    1    2    3    4    5       I    II    III    IV 

ABCD  abed         123456789     10          I     II     III     IV    V     VI 


FIG.  116  A.     GOTHIC  TYPE  FOR  LETTERING  DRAWINGS. 
(From  Optic  Projection). 

§  304.  White  letters  for  black  background.  —  The  white  letters, 
words,  or  numerals  are  most  easily  procured  by  photography.  The 
letters,  words,  etc.,  are  printed  on  tissue  paper.  This  is  used  as  a 
negative  by  placing  it  face  down  on  a  glass  plate  and  in  a  printing 
frame.  Use  some  developing  paper  like  Cyco,  Velox,  etc.,  of  the  con- 
trast variety.  Print  as  for  any  negative  and  develop  with  a  contrast 
developer  so  that  the  whites  and  blacks  will  be  perfect.  The  white 


CH.  VJj 


CLASS  DEMONSTRATIONS 


193 


letters,  etc.,  are  then  cut  out  and  pasted  on  the  drawing  as  described 
above.  This  photographic  paper  is  rather  thick  and  will  show  a 
white  edge  where  it  is  cut.  Blacken  the  white  edges  of  the  letters  or 
words  with  India  ink  after  the  letters  are  stuck  in  place  (fig.  106). 


24  Point  Type    A  a 

123456789  10 

1 

18  Point  Type  A  R  S  2  3  4 

2 

12  Point  Type    ABCabc    1234 

10  Point  Type     ABC     a  b  c    12345 

24  Point  Type    A  a 

123456789  10 

4 

18  Point  Type  ARS  2  34 

4 

12  Point  T,p«    A  B  C  •  b  c    1234 

10  P«4«t  T,g«     »  B  C     •»<    ItS 

FIG.  116  B.    THE  GOTHIC  TYPE  IN  FIG.  116  A.  REDUCED  TO  ONE-HALF  AND  TO 
ONE-FOURTH  NATURAL  SIZE. 

(From  Optic  Projection). 

CLASS  DEMONSTRATIONS 

§  305.  Demonstration  microscopes.  —  Ever  since  the  microscope 
was  invented  physicians  and  naturalists  have  made  the  greatest  use 
of  it  for  demonstration  purposes.  It  was  a  favorite  expression  of 
the  older  writers  that  the  instrument  had  created  a  new  world  of 
the  minute.  Naturally  in  the  beginning  each  person  used  the  instru- 
ment for  himself  as  with  the  simple  microscopes  of  Roger  Bacon. 


194  CLASS  DEMONSTRATIONS  [Cn.  VI 

However,  soon  after  the  invention  of  the  compound  microscope 
Kepler  and  Scheiner  discovered  the  way  to  get  projection  picturesr 
and  these  have  been  much  used  for  demonstrating  to  groups  of  people 
the  enlarged  screen  pictures. 

Recently  the  powerful  lime  and  electric  lights  have  made  it  possible 
to  carry  on  these  demonstrations  to  an  extent  beyond  the  hopes  of 
the  earlier  workers;  and  have  put  facilities  for  helping  students  into 
the  hands  of  the  teacher  which  are  beyond  estimation  in  value. 
Still  for  many  things  and  for  many  persons  having  charge  of  large 
classes  the  individual  simple  or  compound  microscope  is  still  and 
always  will  be  much  used. 

DEMONSTRATION  MICROSCOPES  AND  INDICATORS 
§  306.  Simple  Microscope.  —  Holding  the  simple  microscope  in 
one  hand  and  the  specimen  in  the  other  has  always  been  used  for 
demonstration,  but  for  class  demonstration  it  is  necessary  to  have 
microscope  and  specimen  together  or  the  part  to  be  observed  by 
the  class  is  frequently  missed.  Originally  blocks  of  various  kinds  to 
hold  both  microscope  and  specimen  were  devised,  but  within  the 
last  few  years  excellent  pieces  of  apparatus  have  been  devised  by 
several  opticians  for  the  purpose.  The  accompanying  figure  shows 
one  of  the  best  forms. 

The  tripod  magnifier  and  various  pocket  magnifiers  are  excellent 
for  the  purpose  (fig.  17-18).  Where  the  microscope  and  object 
should  be  held  in  a  fixed  position  the  focusing  stand  for  the  simple 
microscope  is  good  (fig.  19). 

§  307.  Compound  demonstration  microscope.  —  This  was  origi- 
nally called  a  clinical  or  pocket  microscope.  It  is  thus  described  by 
Mayall  in  his  Cantor  Lectures  on  the  history  of  the  microscope: 
"A  small  microscope  was  devised  by  Tolles  for  clinical  purposes 
which  seems  to  me  so  good  in  every  way  that  I  must  ask  special 
attention  for  it.  The  objective  is  screwed  into  a  sliding  tube,  and 
for  roughly  focusing  the  sliding  motion  suffices;  for  fine  adjustment, 
the  sheath  is  made  to  turn  on  a  fine  screw  thread  on  a  cylindrical 
tube,  which  serves  also  as  a  socket  carrier  for  the  stage.  The  com- 
pound microscope  is  here  reduced  to  the  simplest  form  I  have  met 


CH.  VI] 


INDICATOR  OCULARS 


195 


with  to  be  a  really  servicable  instrument  for  the  purpose  in  view; 
and  the  mechanism  is  of  thoroughly  substantial  character.  I  com- 
mend this  model  to  the  notice  of  our  opticians." 

Since  its  introduction  by  Tolles  many  opticians  have  produced 
excellent  demonstration  microscopes  of  this  type,  but  most  of  them 
have  not  preserved  a  special 
mechanism  for  fine  adjustment. 
With  it  one  can  demonstrate  with 
an  objective  of  6  mm.  satisfac- 
torily. It  has  a  lock,  so  that 
once  the  specimen  is  in  the  right 
position  and  the  instrument  fo- 
cused it  may  be  passed  around 
the  class.  For  observation  it  is 
only  necessary  for  each  student 
to  point  the  microscope  toward  a 
window  or  a  lamp. 

A  modification  of  this  clinical 
microscope  was  made  by  Zent- 
mayer  in  which  the  microscope 
was  mounted  on  a  board,  and  a 
lamp  for  illuminating  the  object 
was  placed  at  the  right  position. 

§  308.  Traveling  Microscope.  — 
For  many  years  the  French  op- 
ticians have  produced  most  ex- 
cellent traveling  microscopes.  The  opticians  of  other  countries 
have  also  brought  out  serviceable  instruments.  For  the  needs  of 
the  pathologist  and  sanitary  inspector  a  microscope  must  possess 
compactness  and  also  the  qualities  which  render  it  usable  for  nearly 
all  the  purposes  required  in  a  laboratory.  This  instrument  is  a 
type  of  much  apparatus  which  has  grown  up  with  the  needs  of 
advancing  knowledge. 

§  309.  Indicator  or  pointer  ocular.  —  This  is  an  ocular  in  which 
a  delicate  pointer  of  some  kind  is  placed  at  the  level  where  the  real 
image  of  the  microscope  is  produced.  It  is  placed  at  the  same  level 


A  B 

Fig.    117   A,    B.     POINTER    OCULAR 

AND  FIELD  WITH  POINTER. 

FIG.  117  A.  POINTER  OR  INDICA- 
TOR OCULAR  WITH  A  CAMEL'S  HAIR 
OP)  STUCK  TO  THE  OCULAR  DIA- 
PHRAGM AND  EXTENDING  OUT  INTO 
THE  OPEN  SPACE  WHERE  THE  REAL 
IMAGE  is  FORMED. 

FIG.  117  B.  THE  MICROSCOPIC 
FIELD  OF  A  BLOOD  PREPARATION 
WITH  THE  POINTER  (P)  DIRECTED 
TOWARD  A  LEUCOCYTE. 


I96  INDICATOR  OCULARS  [Cn.  VI 

as  the  ocular  micrometer,  and  the  pointer  like  the  micrometer  is 
magnified  with  the  real  image  and  appears  as  a  part  of  the  projected 
image  (fig.  1 1 7  B) .  By  rotating  the  ocular  or  the  pointer  any  part 
of  the  real  image  may  be  pointed  out  as  one  uses  a  pointer  on  a  wall 
or  blackboard  diagram.  By  means  of  the  indicator  eye-piece  one 


FIG.  118  A.    RING  AROUND  ONE  OF  THE  SECTIONS  OF  A  SERIES  FOR  DEMONSTRAT- 
ING SOME  ORGAN  ESPECIALLY  WELL. 

FIG.  118  B      A  MICROSCOPIC  PREPARATION  WITH  A  RING  AROUND  A  SMALL  PART 
TO  SHOW  THE  POSITION  OF  SOME  STRUCTURAL  FEATURE. 

can  be  certain  that  the  student  sees  the  desired  object,  and  is  not 
confused  by  the  multitude  of  other  things  present  in  the  field.  This 
device  has  been  invented  many  times.  It  illustrates  well  the 'adage: 
"Necessity  is  the  mother  of  invention,"  for  what  teacher  has  not 
been  in  despair  many  times  when  trying  to  make  a  student  see  a 
definite  object  and  neglect  the  numerous  other  objects  in  the  field  ? 
So  far  as  the  writer  has  been  able  to  learn,  Quekett  was  the  first  to 
introduce  an  indicator  ocular  with  a  metal  pointer  which  was  ad- 
justable and  could  be  turned  to  any  part  of  the  field  or  wholly  out 
of  the  field. 

It  is  not  known  who  adopted  the  simple  device  of  putting  a  fine 
hair  on  the  diaphragm  of  the  ocular,  as  shown  in  fig.  117.  This  may 
be  done  with  any  ocular,  positive  or  negative.  One  may  use  a  little 
mucilage,  Canada  balsam,  or  any  other  cement  to  stick  the  hair  on 
the  upper  face  of  the  diaphragm  so  that  it  projects  about  halfway 
across  the  opening.  When  the  eye-lens  of  the  Huygenian  ocular  is 
screwed  back  in  place  the  hair  should  be  in  focus.  If  it  is  not,  screw 
the  eye-lens  out  a  little  and  look  again.  If  it  is  not  now  sharp,  the 
hair  is  a  little  too  high  and  should  be  depressed  a  little.  If  it  is  less 
distinct  on  screwing  out  the  ocular  it  is  too  low  and  should  be 


CH.  VI]  THE  PROJECTION  MICROSCOPE  197 

elevated.     One  can  soon  get  it  in  exact  focus.    Of  course  it  may 
be  removed  at  any  time. 

§  310.  Marking  the  position  of  objects.  —  In  order  that  one 
may  prepare  a  demonstration  easily  and  certainly  in  a  short  time 
the  specimens  to  be  shown  must  be  marked  in  some  way.  An  efficient 
and  simple  method  is  to  put  rings  of  black  or  colored  shellac  around 
the  part  to  be  demonstrated.  For  this  the  Marker  (fig.  59-60)  is 
employed.  For  temporary  marking  an  ink  line  may  be  put  on  with 
a  pen;  or  a  glass  pencil  may  be  used. 

THE  PROJECTION  MICROSCOPE 

311.  Projection  Microscope.  —  One  of  the  most  useful  and 
satisfactory  means  at  the  disposal  of  the  teacher  of  Microscopic 
Anatomy  and  Embryology  for  class  demonstrations  is  the  Projec- 
tion Microscope.  With  it  he  can  show  hundreds  of  students 
as  well  as  one,  the  objects  which  come  within  the  range  of  the 
instrument. 

It  is  far  more  satisfactory  than  microscopic  demonstrations,  for 
with  the  projection  microscope  the  teacher  can  point  out  on  the 
screen  the  structural  features  and  organs  which  he  wishes  to  demon- 
strate, and  he  can  thus  be  certain  that  the  students  know  exactly 
what  is  to  be  studied.  Unless  one  employs  a  pointer  ocular  (fig.  117), 
there  is  no  certainty  that  the  student  selects  from  the  multitude  of 
things  in  the  microscopic  field  the  one  which  is  meant  by  the  teacher. 
Like  all  other  means,  however,  the  projection  microscope  is  limited. 
With  it  one  can  show  organs  both  adult  and  embryonic,  and  the 
general  morphology.  For  the  accurate  demonstration  of  cells  and 
cell  structure  the  microscope  itself  must  be  used.  As  a  general 
statement  concerning  the  use  of  the  projection  microscope  for  demon- 
stration purposes,  it  may  be  said  that  it  is  entirely  satisfactory  for 
objects  and  details  which  show  under  the  microscope  with  objectives 
up  to  1 6  mm.  equivalent  focus.  For  objects  and  details  requiring 
objectives  higher  than  16  mm.  focus  in  ordinary  microscopic  ob- 
servations, the  projection  microscope  is  unsatisfactory  with  large 
classes. 


i98 


THE  PROJECTION   MICROSCOPE 


[CH.  VI 


With  small  classes  (10  or  15)  where  the  screen  distance  can  be 
reduced  to  about  one  meter  demonstrations  with  oil  immersion 
objectives  are  satisfactory.  However,  when  the  finest  details  of 


d  cJeS 


Fig.  10 


FIG.  119.  DIAGRAM  OF  ADAMS'  SOLAR  MICROSCOPE.  THIS  ILLUSTRATES  WELL 
THE  ADVANTAGE  or  SOME  FORM  OF  PROJECTION  MICROSCOPE  FOR  DEMONSTRA- 
TION PURPOSES. 

structure  are  to  be  seen  most  successfully  under  high  powers,  each 
individual  must  look  into  a  microscope  for  himself  and  attend  to  all 
the  finer  adjustment  and  lighting. 


CONDUCT  OF  A  DEMONSTRATION  WITH  THE  PROJECTION 
MICROSCOPE 

§  312.  Preparedness.  —  From  the  great  difficulty  in  making 
really  good  projection  demonstrations  with  the  microscope  the  prepa- 
ration should  be  thorough.  The  following  are  some  of  the  most 
important  things  to  look  after: 

(1)  If  any  of  the  objectives  used  are  of  the  photographic  type 
and  have  an  iris  diaphragm,  that  should  be  opened   to   the  fullest 
possible  extent. 

(2)  The  microscopic  slides  to  be  used  should  be  in  order  so  that 
they  can  be  easily  grasped. 


CH.  VI]  THE  PROJECTION  MICROSCOPE  199 

(3)  If  the  slides  have  many  sections  upon  them,  as  in  a  series, 
then  the  slide  should  be  masked  by  putting  some  orange  paper  over 
the  cover  glass  with  openings  for  the  sections  to  be  shown;  then 
these  can  be  found  quickly  and  with  certainty  (fig.  120,  §  3i2a). 

(4)  Indicate  in  some  way  which  edge  of  the  slide  should  be  up. 
This  will  save  time,  and  add  to  the  respect  for  the  exhibition. 


FIG.  1 20.     SLIDE  TRAY  WITH  MASKED  PREPARATIONS  TO  BE  USED  IN  A  DEMON- 
STRATION. 

(From  Optic  Projection). 

(5)  It  is  often  a  great  help  to  have  stated  on  the  preparation  the 
objectives  best  adapted  to  bring  out  the  special  feature  desired. 

(6)  For  holding  the  specimens,  a  slide  tray  may  be  used  (fig.  120) 
or  one  of  the  slide  boxes.     In  any  case  they  must  be  so  that  the 
slides  can  be  easily  grasped. 

(7)  It  is  for  many  lecturers  easier  to  manipulate  the  projection 
microscope  themselves  and  to  use  a  pointer  held  out  in  the  cone  of 
light.      The  pointer  appears  as  sharply  as  when  put  on  the  screen. 


2OO 


THE  PROJECTION  MICROSCOPE 


[CH.  VI 


(8)  For  all  but  the  highest  powers  a  substage  condenser  is  not 
needed;   and  one  can  light  objects  up  to  50  or  60  mm.  in  diameter  if 
the  object  is  placed  in  the  right  position  in  the  cone  of  light  (fig.  124). 

(9)  For  objectives  of  higher  power  than  4  mm.  a  substage  con- 
denser should  be  used,  and  if  an  ocular  is  used  as  well  as  an  objective 
then  the  substage  condenser  is  advantageous  for  powers  above  8  mm. 
equivalent  focus.     For  lighting  see  §  104,  107,  352. 


PIG.  121.    ILLUMINATING   OBJECTS    OF   VARIOUS    SIZES    IN    MICRO- PROJECTION 
WITH  THE  MAIN  CONDENSER  ONLY. 

(From  Optic  Projection). 

The  object  must  be  put  in  the  cone  of  light  at  a  point  where  it  will  be  fully 
illuminated. 

For  high  powers  it  will  be  at  or  very  near  the  focus  (/).  For  larger  objects  and 
low  powers  at  2  or  3,  or  even  closer  to  the  condenser  face. 

Arc  Supply    The  right-angled  carbons  of  the  arc  lamp. 

L\  LZ    The  first  and  second  elements  of  the  triple  condenser. 

Water  Cell  The  water  cell  for  absorbing  radiant  heat.  It  is  in  the  parallel 
beam  between  the  first  and  second  elements  of  the  condenser. 

Axis    The  principal  optic  axis  on  which  all  the  parts  are  centered. 

(10)  One  of  the  most  important  points  is  to  have  a  very  white 
screen.  A  cloth  or  wall  screen  painted  with  Artist's  Scenic  White 
gives  a  very  perfect  screen  which  does  not  yellow  with  age,  and  its 
primitive  whiteness  is  restored  by  an  occasional  coat  of  fresh  white. 
Semi-mirror  screens  are  successful  only  in  narrow  rooms. 

For  short  screen  distances  (i  or  2  meter  screen  distances)  white 
cardboard  or  a  sheet  of  very  white  bristol  board  gives  excellent 
results. 

The  apparatus,  in  contrast  to  the  screen,  should  be  dull  black. 

§  312a.  Masks  for  demonstration  slides.  — The  paper  to  use  should  allow 
the  red  and  orange  to  pass,  but  cut  off  the  green-blue  end  of  the  spectrum.  An 


CH.  VI]  THE  PROJECTION  MICROSCOPE  201 

excellent  masking  paper  can  be  prepared  by  soaking  white  paper  in  a  saturated 
aqueous  solution  of  Orange  G  for  10  to  15  minutes,  and  then  hanging  it  up  until 
dry.  The  paper  is  then  cut  into  pieces  the  size  of  the  cover-glass.  Holes  are  made 
opposite  the  sections  to  be  demonstrated  (fig.  120),  and  the  paper  pasted  to  the 
cover-glass.  It  is  put  on  the  cover-glass  and  not  on  the  under  side  of  the  slide  be- 
cause if  on  the  slide  it  would  prevent  the  conduction  of  the  heat  absorbed.  If  the 
slide  rests  against  the  metal  or  glass  stage  the  absorbed  heat  is  largely  conducted 
away  by  the  stage  water  cell  or  the  metal  stage. 

If  one  wishes  to  remove  the  mask  from  the  cover  the  safest  way  is  to  place  a 
piece  of  wet  blotting  paper  on  the  mask  to  soften  it.  It  can  then  be  easily  re- 
moved and  the  cover-glass  cleaned  with  a  moist  cloth. 

§  313.     Objectives,  oculars,  and  amplifiers  to  use  in  projection.  - 

Objectives  of  the  photographic  type  from  100  to  16  mm.  equivalent 
focus  are  unexcelled.  They  should  not  be  used  with  oculars.  Of 
ordinary  objectives,  all  powers  can  be  used,  but  for  demonstrations 
before  large  classes  a  4  mm.  is  the  highest  power  found  really  satis- 
factory. The  ones  most  used  are  of  16,  10,  8  mm.  focus.  Oculars 
of  the  projection  or  the  ordinary  type  answer  well.  If  the  projection 
type  is  used  one  must  rotate  the  eye-lens  until  there  is  a  sharp  image 
of  the  diaphragm  on  the  screen  to  get  the  best  image  of  the  object. 

If  one  is  to  use  an  amplifier  with  the  objective,  but  no  ocular,  the 
increase  in  size  should  not  be  over  1.5  to  2.5.  beyond  that  given  by 
the  objective.  For  example  if  the  screen  image  with  the  objective 
alone  were  50x5  diameters,  the  amplifier  should  not  enlarge  it  beyond 
750  or  1000. 

The  ones  used  by  the  author  are  for  the  large  tube  of  the  micro- 
scope (fig.  112)  and  are  nearly  5  cm.  in  diameter  to  fit  the  microscope 
tube.  Their  virtual  foci  are  20  and  10  cm.  (5  and  10  diopter  con- 
caves) . 

§  314.  Centering  the  optical  parts  on  one  axis.  —  This  is  one  of 
the  most  important  procedures  of  all  and  no  good  projection  can  be 
accomplished  without  it.  The  easiest  way  is  to  first  arrange  the 
crater  of  the  arc  lamp,  the  central  point  of  the  large  condenser,  and 
the  microscope  objective  all  at  the  same  height  from  the  base  board 
(fig.  in).  If  then  the  lamp  is  turned  on  and  the  objective  placed 
in  the  focus  of  the  main  condenser  cone,  the  image  of  the  crater  of 
the  arc  lamp  should  be  formed  on  the  end  of  the  objective,  the  brightest 
part  on  the  front  lens.  If  the  image  is  to  one  side,  above  or  below, 
then  the  microscope  should  be  raised,  or  lowered.  After  being  once 


2O2 


THE  PROJECTION  MICROSCOPE 


[CH.  VI 


carefully  centered,  the  centering  will  vary  slightly  with  the  burning 
of  the  carbons.  To  compensate  for  this  there  must  be  fine  adjust- 
ments to  raise  and  lower  the  carbons  and  to  move  them  from  side  to 


w 

? 

Outlet 

Box 

Supply  Wires 


Arc 
Supply 


FIG.  122.     PROJECTION  APPARATUS  SHOWING  THE  PARTS  AND  THE  WIRING  FOR 

AN  ARC  LAMP. 

(From  Optic  Projection). 

The  Objective,  Condenser,  and  Arc  Lamp  are  on  separate  blocks  which  move 
independently  along  the  optical  bench  (fig.  131). 

c     Center  of  the  objectives  where  the  rays  from  the  condenser  should  cross. 

i,  2  The  first  and  second  elements  of  the  three  lens  condenser  with  a  water 
cell  for  absorbing  radiant  heat  between  the  lenses. 

V    The  ventilating  hood  of  the  lamp  house. 

LA ,  VA  The  mechanism  for  fine  adjustment  of  the  arc  lamp  to  the  sides  and 
vertically.  These  are  a  necessity  for  projecting  with  the  microscope,  otherwise 
the  crater  cannot  be  kept  centered. 

FS    The  fine  adjustments  for  the  two  carbons. 

PWR  Separable  attachment  for  the  wires  from  the  outlet  box  to  the  table 
switch. 

W\     Wire  from  the  table  switch  to  the  upper  carbon. 

W2     Wire  from  the  table  switch  to  the  rheostat. 

Wj    Wire  from  the  rheostat  to  the  lower  carbon. 

side.     No  good  projection  can  take  place  unless  the  full  cone  of 
light  shines  upon  the  end  of  the  objective. 

To  get  the  very  best  effect  in  the  easiest  way  there  should  be  a  dull 
black  shield  over  the  end  of  the  objective  (fig.  123)  so  that  the  image  of 
the  crater  can  be  seen  without  hurting  the  eyes.  When  the  crater  is 
focused  on  the  end  of  the  objective  the  specimen  is  moved  up  until 


CH.  VI] 


THE  PROJECTION  MICROSCOPE 


203 


it  is  in  focus,  the  objective  not  being  moved.  Of  course  this  means 
that  the  stage  must  be  separately  movable  (fig.  109).  See  also 
§296. 

§  315.     Demonstrations  with  a  vertical  projection  microscope.  - 
Many  specimens  must  be  mounted  in  liquids  and  cannot  be  set  in  a 
vertical  position;  therefore  the  microscope  must  be  vertical  and  the 
object  remain  horizontal.     In  such  a  case  project  the  light  from 


FIG.  123  A,  B.    METAL  HOOD  OVER  THE  OBJECTIVE  TO  AID  IN  CENTERING  THE 

LIGHT. 

(From  Optic  Projection). 

A     Longitudinal  section  of  the  objective  to  show  the  metal  hood. 

B  End  view  of  the  objective  with  the  crater  of  the  arc  lamp  directly  in  the 
center  at  the  left  and  to  one  side  of  the  center  at  the  right.  The  adjustments, 
VA,  LA  in  fig.  122  are  to  enable  one  to  center  the  light  easily. 

the  large  condenser  (fig.  in)  or  from  the  small  arc  lamp  (fig.  49) 
upon  the  mirror  of  the  microscope  and  reflect  it  directly  upward, 
and  then  use  a  mirror  or  prism  to  change  the  direction  from  vertical 
to  horizontal.  (See  fig.  109,  114,  to  recall  how  the  beam  is  changed 
in  direction  90°.) 

A  most  striking  preparation  is  one  of  the  hay  infusion  (§211) 
projected  upon  the  screen.  A  water  immersion  objective  of  2  to  3 
mm.  equivalent  focus  is  excellent  for  projecting  such  preparations. 


DEMONSTRATION  LANTERN  AND  TABLE  FOR  ARTIFICIAL  DAYLIGHT 

§  316.  Special  microscopic  demonstrations.  —  As  stated  above, 
if  one  is  to  see  the  finest  details  of  structure  there  is  no  satisfactory 
way  but  to  look  into  the  microscope  direct.  There  is  also  in  every 
laboratory  for  microscopic  work  considerable  waste  space  if  depen- 


204 


KINGSBURY'S  DEMONSTRATION  TABLE 


[CH.  VI 


dence  is  put  upon  daylight.     If  artificial  light  is  used  regularly  the 
method  here  given  is  also  applicable. 

The  main  points  for  this  kind  of  demonstration  were  worked  out 
by  Dr.  B.  F.  Kingsbury  for  his  laboratory  of  Histolog}'  and  Em- 
bryology. 


FIG.  124  A,  B.     KINGSBURY'S  DEMONSTRATION  TABLE  WITH  ARTIFICIAL  DAY- 
LIGHT.    (ABOUT  ^  NATURAL  SIZE). 

(From  the  Anatomical  Record,  June,  1916). 

T  Top  of  the  metal  tube  and  the  separable  attachment  plug.  This  tube 
reaches  about  2  meters  above  the  floor  so  that  the  supply  cable  will  be  out  of  the 
way. 

N  S    The  single  250  watt  mazda  lamp  with  its  metal  support. 

i,  2,  3,  4,  5,  6,  7,  8  The  shields  (SH)  with  a  disc  of  daylight  glass  (a)  in  each 
at  the  level  of  the  microscope  mirror. 

A  round  top  demonstration  table  of  a  size  for  8  microscopes  is 
made  and  in  the  middle  a  single  mazda  lamp  of  200  or  250  watts  is  in- 
stalled (fig.  124  A).  Around  this  lamp  are  8  shields,  each  containing 
a  piece  of  daylight  glass. 

With  this  arrangement  8  microscopes  can  be  used  at  once  (fig. 
1 24  B)  and  the  light  is  sufficient  to  enable  the  student  to  use  all  powers 
of  the  microscope  up  to  the  highest  oil  immersion.  This  method 
of  demonstration  has  already  been  in  use  during  the  entire  college 
year  of  1915-1916  and  has  proved  successful  beyond  expectation. 


CH.  VI] 


LABORATORY   TABLK 


205 


§  316a.  Line  drawings  on  the  back  of  photographs.  —  Repeated  use  of  the 
method  for  obtaining  line  drawings  on  the  back  of  photographic  prints  during  the 
last  four  years  has  demonstrated  its  great  usefulness.  The  artist  or  the  amateur  has 
the  advantage  of  the  photographic  image  with  its  correct  proportions  and  perspec- 
tive, and  is  in  no  way  hindered  from  adding  free  hand  additional  details,  or  artistic 
touches.  The  enormous  advantage  of  line  cuts  over  half  tones  becomes  apparent 
with  increasing  emphasis  with  every  book  or  scientific  article  published  with  half- 
tone illustrations  with  their  vagueness.  Photography  is  a  good  helper,  but  should 
not  be  made  the  master  in  getting  scientific  pictures. 


FIG.  125.     Two  MICROSCOPES  AND  A  CHALET  LAMP  ox  A  LABORATORY  TABLE. 
(About  one  ninth  natural  size.) 

The  Chalet  microscope  lamp  with  two  windows  (fig.  37)  serves  well  for  two 
observers  on  opposite  sides  of  the  same  table,  or  two  tables  may  be  placed  side 
by  side  and  the  lamp  rested  partly  on  each. 


COLLATERAL  READING  FOR  CHAPTER  YL 

ATWELL,  W.  J.  —  On  the  conversion  of  a  photograph  into  a  line  drawing.  Anat. 
Record,  Vol.  10,  pp.  39-41.  The  lines  are  rgade  on  the  face  of  the  photograph, 
then  the  photographic  image  is  bleached  out  by  means  of  hypo  and  cyanide. 

COMSTOCK,  J.  H.  —  The  Wings  of  Insects,  1918.  Blue  prints  were  made  and  the 
India  ink  lines  made  on  their  face.  The  blue  was  then  bleached  with  po- 
tassium oxalate,  etc. 

GAGE,  S.  H.  AND  H.  P.,  Optic  Projection,  Ch.  X. 

HARDESTY  AND  LEE.     Laboratory  drawing. 


CHAPTER  VII 

PHOTOGRAPHING  EMBRYOS  AND   SMALL  ANIMALS;    PHOTO- 
GRAPHIC ENLARGEMENTS;    PHOTOGRAPHING  WITH  THE 
MICROSCOPE 

§  325.     Apparatus  and  material  for  Chapter  VII.  — 

1.  Photographic     cameras,     hori-  8.    Daylight     lantern     and    other 
zontal  and  vertical  (§  327-328).  ^  lights  (§  345,  373~374)- 

2.  Focusing     stand     for     vertical  9.    Stage  micrometer  (§  350). 
camera  (§  333).  10.    Projection   apparatus   (§  359- 

3.  Focusing  glass  (§  334).  363) 

4.  Objectives    for    photographing  n.    Photographic     chemicals 
embryos  (§  335).  (§  377). 

5.  Negative  records  (§  337,  358).  12.    Plates     and     printing,  paper 

6.  Photo-micrographic     camera  (§362,379-380). 

(§  338,  34o)-  13-    Color  screens    (§  366-368). 

7.  Microscope  oculars  and  objec-  14.    Microspectroscope  (§  371). 
tives  (§  342,  343,  355).  15.    Dark-room  light   (§  378). 

16.    Printing    frames    (§  362-363). 

PHOTOGRAPHY 

§  326.  From  the  beginning  of  the  art  of  photography  scientific 
men  have  used  it  to  paint  for  them  the  forms  in  nature  and  the  com- 
plex structures  found  in  the  physical  and  the  biological  world;  and 
it  has  been  so  good  a  servant  that  it  is  more  and  more  called  into 
requisition  to  delineate  all  the  phenomena  as  well  as  the  forms  of 
nature  and  art.  This  is  especially  true  now  that  successful  methods 
of  color  photography  have  become  available. 

§  327.  Photography  with  a  horizontal  camera.  —  The  most  con- 
venient position  for  the  camera  obscura  is  the  horizontal  one  (fig. 
1 08)  and  for  most  of  the  photography  actually  done  it  is  very  easy 
to  arrange  the  objects  to  be  photographed  in  a  vertical  position; 
but  for  much  of  the  photography  of  science  it  is  very  convenient  to  use 
a  vertical  camera,  leaving  the  objects  in  a  horizontal  position.  With 
objects  in  liquids  this  is  a  practical  necessity. 

206 


CH.  VII]       PHOTOGRAPHY  WITH  A  VERTICAL  CAMERA  207 

§  328.  Photography  with  a  vertical  camera.  —  The  object  can  be 
left  horizontal  as  well  as  the  camera  by  the  use  of  a  mirror  or  totally 
reflecting  prism,  but  this  gives  the  inversion  of  a  plane  mirror,  and 
as  shown  in  §  282  it  will  render  the  image  erect  on  the  film  side  of  the 
negative,  but  when  the  negative  is  printed  the  image  will  be  inverted. 
To  meet  all  the  difficulties  the  object  may  be  left  in  a  horizontal 
position  and  the  camera  made  vertical  (fig.  126). 

Since  1879  such  a  camera  has  been  in  use  in  the  Anatomical  Depart- 
ment of  Cornell  University  for  photographing  all  kinds  of  specimens; 
among  these,  fresh  brains  and  hardened  brains  have  been  photo- 
graphed without  the  slightest  injury  to  them.  Furthermore,  as 
many  specimens  are  so  delicate  that  they  will  not  support  their  own 
weight,  they  may  be  photographed  under  alcohol  or  water  with  a 
vertical  camera  and  the  result  will  be  satisfactory  as  a  photograph 
and  harmless  to  the  specimen  (§  328a). 

A  great  field  is  also  open  for  obtaining  life-like  portraits  of  water 
animals.  Chloretoned  or  etherized  animals  are  put  into  a  vessel  of 
water  with  a  contrasting  background  and  arranged  as  desired,  then 
photographed.  Fins  have  something  of  their  natural  appearance 
and  gills  of  branchiate  salamanders  float  out  in  the  water  in  a  natural 
way.  In  case  the  fish  tends  to  float  in  the  water  a  little  mercury 
injected  into  the  abdomen  or  intestine  will  serve  as  ballast.  The 
photographs  obtainable  in  water  are  almost  if  not  quite  as  sharp  as 
those  made  in  air.  Even  the  corrugations  on  the  scales  of  such  fishes 
as  the  sucker  (Catostomus  teres)  show  with  great  clearness. 

While  the  use  of  photography  diminishes  the  labor  of  artists  about 
one-half,  it  increases  that  of  the  preparator;  and  herein  lies  one  of 
its  chief  merits.  The  photographs  being  exact  images  of  the  prepara- 
tions, the  tendency  will  be  to  make  them  with  greater  care  and  deli- 
cacy, and  the  result  will  be  less  imagination  and  more  reality  in 
published  scientific  figures;  and  the  objects  prepared  with  such 
care  will  be  preserved  for  future  reference. 

In  the  use  of  photography  for  figures  sevecal  considerations  arise: 
(i)  The  avoidance  of  distortion;  (2)  The  adjustment  of  the  camera 
to  obtain  an  image  of  the  desired  size;  (3)  Focusing;  (4)  Lighting 
and  arranging  the  object. 


208  PHOTOGRAPHY  WITH  A  VERTICAL   CAMERA      [Cn.  VII 

(i)  While  the  camera  delineates  rapidly,  the  image  is  liable  to 
distortion.  I  believe  opticians  are  agreed  that,  in  order  to  obtain 
correct  photographic  images,  the  objective  must  be  properly  made, 
and  the  plane  of  the  object  must  be  parallel  to  the  plane  of  the  ground- 
glass.  Furthermore,  as  most  of  the  objects  in  natural  history  have 
not  plane  surfaces,  but  are  situated  in  several  planes  at  different  levels, 
the  whole  object  may  be  made  distinct  by  using  a  long  focus  objective 
and  a  small  diaphragm. 

§  328a.  Papers  on  this  subject  were  given  by  the  writer  at  the  meeting  of  the 
American  Association  for  the  Advancement  of  Science  in  1879,  and  at  the  meeting 
of  the  Society  of  Naturalists  of  the  eastern  United  States  in  1883;  and  in  Science 
Vol.  Ill,  pp.  443,  444-  • 

§  329.  Scale  of  photographs.  —  It  is  desirable  to  make  all  photo- 
graphs at  some  definite  scale.  To  do  this  without  much  waste  of 
time  the  camera  should  be  calibrated  for  each  objective  that  is  to  be 
used.  This  is  easily  accomplished  by  using  a  metric  scale  like  that 
shown  in  fig.  104.  By  lengthening  and  shortening  the  bellows  of 
the  camera  so  that  the  image  distance  is  greater  and  less  one  can 
get  the  exact  position  for  a  group  of  magnifications  and  reductions. 
If  the  length  of  the  bellows  is  noted  for  each  size,  and  the  distance, 
of  the  objective  from  the  object  when  the  focus  is  good  is  also  noted 
one  can  arrange  the  camera  very  quickly  for  any  special  size  which 
may  be  desired.  The  sizes  found  very  useful  by  the  author  are: 
^;  i;  J;  i;  i;  2;  2-5;  4;  5-  For  magnifications  above  5  it  is  better 
to  make  a  negative  natural  size  (xi)  and  then  make  an  enlargement  of 
this,  as  explained  in  §  359. 

The  vertical  camera  shown  in  fig.  126  has  the  supporting  rod  gradu- 
ated in  centimeters  and  half  centimeters.  After  the  extension  of 
the  camera  for  any  size  has  been  once  determined,  it  is  easily  made 
the  same  at  some  future  time. 

§  330.  Magnification  rod  for  the  camera.  —  Objects  vary  so  much 
in  thickness  that  the  focusing  range  of  the  camera  should  be  consid- 
erable. With  the  ordinary  camera  there  is  usually  no  provision  for 
moving  the  camera  as  a  whole  for  focusing.  With  the  vertical  camera 
shown  in  fig.  126,  where  both  ends  of  the  camera  must  be  clamped,  it 
is  difficult  to  focus  over  a  large  range  and  keep  the  length  of  camera 


CH.  VII]       PHOTOGRAPHY  WITH  A   VERTICAL   CAMERA  209 

needed  for  the  desired  magnification  or  reduction.  For  this  reason 
the  same  device  was  applied  to  it  as  to  the  original  vertical  camera 
of  1879,  viz.,  a  rod  passing  from  end  to  end  of  the  camera,  fixed  at 
one  end  and  clamped  at  the  other.  When  the  camera  is  extended 
the  exact  amount  required  for  the  size  in  a  given  case  the  clamp  is 
fixed  so  that  the  length  of  the  camera  cannot  be  changed,  then  the 
whole  camera  may  be  moved  for  focusing  without  any  danger  of 
varying  the  magnification.  This  device  saves  a  great  deal  of  time 
and  keeps  one  good-natured.  In  the  original  camera  of  1879,  the 
rod  was  graduated  in  centimeters.  This  of  course  helps  to  give  the 
proper  extension  with  the  least  outlay  of  trouble.  In  fig.  126  the  ver- 
tical supporting  rod  is  graduated  in  centimeters  and  half  centimeters. 

§  331.  Lighting  for  the  vertical  camera.  —  The  object  should  be 
so  arranged  that  all  the  details  come  out  with  the  greatest  distinct- 
ness.. As  the  light  must  be  largely  from  the  side  it  is  often  necessary 
to  put  a  piece  of  white  blotting  paper  or  cardboard  on  the  side  of 
the  specimen  opposite  the  window.  Occasionally  for  lighting  up 
deep  cavities  it  is  a  great  advantage  to  use  a  mirror  and  reflect  sun- 
light into  the  cavities  for  a  part  of  the  exposure. 

Great  care  must  be  taken  in  selecting  a  suitable  background  so 
that  the  specimen  will  stand  out  clearly  and  not  be  merged  into  the 
background. 

When  a  white  background  is  used,  the  shadow  of  the  specimen  is 
often  very  troublesome,  and  to  distinguish  the  outline  of  the  object 
W.  E.  Rumsey  (Canadian  Entomologist,  1896,  p.  84)  hit  upon  the 
plan  of  placing  the  object  on  a  glass  plate  and  putting  the  back- 
ground on  a  stage  below  (fig.  126).  A  background  on  the 
lower  stage  does  away  with  the  confusion.  If  daylight  is  not 
available  excellent  photographs  can  be  obtained  with  mazda  lamps 
with  metallic  or  white  reflectors  to  direct  the  light.  It  is  usually 
better  to  employ  two  portable  lamps  and  arrange  them  so  that  the 
shadows  will  not  be  too  prominent. 

§  332.  —  Photographing  embryos,  small   animals,   and   organs.  — 
The  camera  shown  in  fig.  126  is  admirably  adapted  for  this,  as  the 
objects,  many  of  them,  must  be  photographed  under  water,  alcohol, 
or  other  liquids. 


210 


FOCUSING  IN  PHOTOGRAPHY 


[CH.  VII 


If  one  has  a  good  place  to  do  the  work  in,  the  light  can  usually  be 
arranged  satisfactorily  with  the  object  in  a  vessel  with  a  proper 
background  in  the  bottom.  If  not,  a  double 
stage  must  be  used,  as  shown  in  fig.  126. 

If  white  embryos  or  other  light  objects 
are  to  be  photographed  a  black  background 
is  best.  This  is  produced  by  using  black 
glass  on  the  bottom  of  the  dish.  Or  if  no 
black  glass  is  available,  some  smooth  paper 
is  coated  with  waterproof  India  ink  and  al- 
lowed to  dry.  This  gives  good  contrast. 

With  a  proper  background  make  sure  that 
the  lighting  is  such  as  to  bring  out  the  de- 
sired details.  Turn  the  object  in  various 
positions  till  the  desired  one  is  found  which 
shows  clearly  the  points  that  are  to  be  em- 
phasized. 

§  333.  Focusing  stand  for  the  vertical  cam- 
era. —  To  hold  the  specimen  and  to  provide 
for  the  finest  focusing,  and  also  some  of  the 
coarse  focusing,  a  modified  microscope  stand 
is  convenient.  It  has  no  tube,  but  two  stages 
are  attached  to  the  support  usually  carrying 
the  tube.  This  then  can  be  raised  and  low- 
ered by  the  coarse  and  by  the  fine  adjust- 


FIG.    126.      VERTICAL 
PHOTOGRAPHIC  CAMERA. 


T  Low  table  50  cm. 
high,  50  cm.  wide,  and  70 
cm.  long. 

with5  vessel1  Sforg  holding    ment>  as  in  focusing  the  microscope,  except 
embryos  and  small  ani-    here  the  stages  move,  the  photographic  ob- 
jective remaining  stationary  (fig.  126).    With 
the  rod  to  hold  the  camera  at  a  fixed  ex- 


mals  to  be  photographed 
under  liquid. 

VC     Vertical    camera 


(See  fig.  107  for, fuller  de- 
scription.) 


tension,   most  of  the  focusing  can  be  ac- 
cusing glass  (fg)  above,    complished  by  sliding  the  whole  camera  up 
and   down  the  vertical  graduated  support 
(fig.  126). 

§  334.  —  Focusing  glass.  —  There  are  two  ways  of  using  this: 
i.   A  clear  screen  is  used  instead  of  aground-glass.     On  this  is  a 
diamond  scratch  in  the  middle.     The   focusing   glass  is  carefully 


CH.  VII] 


FOCUSING  IN  PHOTOGRAPHY 


211 


focused  on  the  central  scratch,  which  must  be  in  the  exact  plane  where 
the  sensitized  photographic  surface  will  be  during  the  exposure.  If 
now  an  object  is  brought  to  an  accurate  focus  at  this  plane,  it  will 
also  be  in  focus  on  the  sensitized  surface  of  the  dry  plate.  Except 
for  aid  in  arranging  the  object  and  for  general  focusing,  the  frosted 
glass  can  be  entirely  omitted,  and  a  ^^^^^^^^^^^^^^^^^^ 
focusing  glass  giving  about  8  to  10 
diameters  magnification  is  set  in  a 
board  which  takes  the  place  of  the 
ordinary  frosted  glass  screen.  This 
is  put  at  the  level  to  bring  the  focus 
exactly  at  the  plane  where  the  sensi- 
tive surface  of  the  negative  is  to  be. 

The  position  of  the  focusing  glass 
is  determined  as  follows: 

The  plate  holder  with  a  clear  glass 
plate  or  a  thin  negative  is  in  the 
holder.  And  on  the  film  side  is  a 
diamond  scratch  or  an  India  ink 
mark  near  the  middle  of  the  face 

usually  occupied  by  the  sensitive  film. 

T,    .  .  ,,  J     The  ground  or  frosted  sur- 

It  is  very  important  that  the  mark    face  of  the  glass. 

should  be  on  the  side  occupied  by     p    2    A  cover-glass  stuck  to  the 

.  frosted  surface  with  Canada  Bal- 

tne  nlm.  sam.     This    renders    the    frosted 

The  scratch  or  ink  mark  is  a  guide    surface  transparent. 
e  -ir  •  i  x     Pencil  mark  in  the  center  of 

for    getting    the    focus    at    the    right     the  focusing  screen  on  the  frosted 
level.     Now  with  a  tripod  or  other     surface  to   serve  as  guide  when 

.  -  r       ,  .  focusing  with  a  magnifier, 

magnifier,  preferably  with  the  mag- 
nifier to  be  used  later,  get  the  image  focused  of  the  metric  scale  and 
its  explanation  or  other  sharp  print  exactly  on  the  surface  where 
the  diamond  or  ink  mark  is.  To  make  sure  that  there  is  no  better 
focus  obtainable  it  is  worth  while  to  make  a  negative  of  the  printed 
matter  used  for  focusing.  On  the  excellence  of  the  focus  determined 
depends  the  excellence  of  all  future  pictures  which  will  be  made.  This 
method  has  the  further  advantage  that  the  focus  level  is  determined 
for  the  plate  holder  and  not  for  a  focusing  screen.  It  is  in  fact  an 


FIG.  127.  GROUND-GLASS  FO- 
CUSING SCREEN  WITH  CLEAR  CEN- 
TER FOR  FINE  FOCUSING. 


212  FOCUSING  IN  PHOTOGRAPHY  [Cn.  VII 

excellent  way  to  check  up  the  similarity  of  level  of  the  ordinary 
focusing  screen  and  the  plate  holder.  Frequently  they -do  not  agree 
closely  enough  for  the  more  exacting  work,  especially  'in  photo- 
micrography. If  the  focus  is  found  to  be  exact  proceed  to  set  the 
focusing  glass  in  a  board  as  follows: 

Have  a  board  of  about  15  mm.  thickness  in  a  frame  like  that  used 
for  the  ordinary  focusing  screen.  Bore  a  hole  in  the  center  in  which 
the  focusing  glass  holder  will  fit  snugly.  Now 
put  the  frame  on  the  focused  camera  and 
slowly  twist  the  focusing  glass  into  the  hole 
until  the  focus  seen  through  it  is  perfect.  If 
nothing  has  changed  in  the  camera  then  this 
focus  should  give  perfect  results  for  any 
future  setting  of  the  camera,  for  the  focus 
will  be  at  the  exact  level  occupied  by  the 
sensitive  surface  of  the  plate.  If  it  is  found 
.FIG.  128.  TRIPOD  perfect  by  trial,  it  is  wise  to  put  some  shellac 
MAGNIFIER  TO  SERVE  Qr  Qther  vamish  around  the  mounting  to  fix 

AS    A    rOCTJSING    OLASb. 

it  firmly  in  place  in  the  wood  so  that  there 

will  be  no  change  in  its  position.  Of  course  any  change  would  re- 
sult in  imperfect,  out-of-focus  negatives. 

This  method  of  focusing  has  the  great  advantage  of  doing  away  with 
all  obstructing  glass.  One  focuses  the  position  of  the  real  image  ex- 
actly as  for  a  compound  microscope  when  a  positive  ocular  (fig.  22)  is 
used.  It  is  an  invaluable  way  for  focusing  in  photo-micrography. 

§  335.  Objectives  and  magnification  for  embryos.  —  It  is  a  good 
plan  to  have  one  picture  of  natural  size  in  each  case,  and  then  if  the 
embryos  or  other  objects  are  very  small,  a  picture  of  5  or  more  times 
natural  size.  And  a  picture  should  go  with  the  embryo  or  object 
throughout  its  entire  career  so  that  the  exact  appearance  before  sec- 
tioning or  dissection  will  be  available. 

Objectives  for  making  photographs  of  from  xi  to  x5  range  from 
50  to  100  mm.  equivalent  focus,  and  they  are  placed  in  the  end  of 
the  camera  as  usual  (fig.  126).  The  larger  the  object  the  longer  should 
be  the  focus  of  the  objective;  then  the  exaggerated  perspective  of 
short  focused  lenses  will  be  avoided. 


CH.  VII]  PHOTOGRAPHY  WITH  DARK-GROUND  ILLUMINATION  213 

§  336.  Photographing  bacterial  cultures.  —  For  the  successful 
photographing  of  these  cultures  dark-ground  illumination  is  employed 
on  the  principle  stated  in  §  117.  That  is,  the  preparation  is  illumi- 
nated with  rays  so  oblique  that  none  can  enter  the  objective  directly. 
These  striking  the  culture  are  reflected  into  the  objective.  The  clear 
gelatin  around  the  growth  or  colonies  does  not  reflect  the  light  and 
therefore  the  space  between  the  colonies  is  dark. 

For  supporting  the  Petri  dishes  a  hole  is  made  in  a  front  board  for 
the  camera.  This  hole  is  slightly  larger  than  the  dish.  Over  it  is 
then  screwed  or  nailed  a  rubber  ring  slightly  smaller  than  the  Petri 
dish.  This  will  stretch  and  receive  the  dish,  and  grasp  it  firmly,  so 
that  it  is  in  no  danger  of  falling  out  when  put  in  a  vertical  position. 
If  the  camera  has  two  divisions  like  the  one  shown,  the  board  with 
the  Petri  dish  is  put  in  the  front  of  the  camera,  and  the  objective  in 
the  middle  division  through  the  side  door.  Otherwise  the  board 
holding  the  Petri  dish  must  be  on  a  separate  support  (fig.  108). 

The  vertical  camera  and  focusing  stand  (fig.  126)  lend  themselves 
admirably  for  this  kind  of  photography.  The  black  background  can 
be  put  on  the  lower  stage  and  the  Petri  dish  or  other  bacterial  culture 
can  be  set  on  a  glass  plate  or  in  a  perforated  board  on  the  upper 
stage.  The  lighting  is  very  easily  accomplished  by  two  portable 
lamps  so  arranged  that  no  light  can  get  directly  from  them  into  the 
objective. 

One  may  use  daylight  by  putting  the  culture  in  a  support  just  out- 
side a  window,  leaving  the  camera  in  the  room.  The  rays  from  the 
sky  are  so  oblique  that  they  do  not  enter  the  objective.  One  must 
use  a  black  non-reflecting  background  some  distance  beyond  the  dish 
as  in  using  artificial  light  (Atkinson). 

In  photographing  bacterial  cultures  in  test-tubes  the  lighting  is 
as  in  the  preceding  section,  but  a  great  difficulty  is  found  in  getting 
good  results  from  the  refraction  and  reflections  of  the  curved  surfaces. 
To  ovecome  this  one  applies  the  principles  discussed  in  §  202,  and 
the  test-tubes  are  immersed  in  a  bath  of  water  or  water  and  glycerin. 
The  bath  must  have  plane  surfaces.  Behind  it  is  the  black  velvet 
screen,  and  the  light  is  in  front,  as  for  the  Petri  dishes.  As  suggested 
by  Spitta,  it  is  well  to  employ  a  bath  sufficiently  thick  in  order  that 


214  PHOTOGRAPHING  WITH  THE  MICROSCOPE         [Cn.  VII 

streak  cultures  may  be  arranged  so  that  the  sloping  surface  will  all 
be  in  focus  at  once  by  inclining  the  test-tube. 

§  337.  Recording  and  storing  negatives.  —  Each  negative  should 
have  a  record  upon  it  written  on  the  film  side  with  India  ink;  then  it 
will  never  get  mixed  up.  For  ease  in  finding  them  there  should  be 
a  record  on  the  containing  envelope  also.  Finally,  it  is  a  good  plan 
to  have  a  card  catalogue  of  one's  negatives.  For  a  form  see  §  358. 

For  storing  negatives  a  good  method,  where  one  does  not  have 
too  many,  is  to  put  them  in  envelopes  and  store  in  boxes  or  drawers 
like  book  catalogue  cards. 

PHOTOGRAPHING  WITH  THE  MICROSCOPE 

§  338.  The  first  pictures  made  on  white  paper  and  white  leather, 
sensitized  by  silver  nitrate,  were  made  by  the  aid  of  a  solar  microscope 
(1802).  The  pictures  were  made  by  Wedgewood  and  Davy,  and  Davy 
says:  "I  have  found  that  images  of  small  objects  produced  by  means 
of  the  solar  microscope  may  be  copied  without  difficulty  on  prepared 
paper."  (§  338a). 

Thus  among  the  very  first  of  the  experiments  in  photography  the 
microscope  was  called  into  requisition.  And  naturally  plants  and 
motionless  objects  were  photographed  in  the  beginnings  of  the  art 
when  the  time  of  exposure  required  was  very  great. 

Although  first  in  the  field,  photo-micrography  has  been  least 
successful  of  the  branches  of  photography.-  This  is  due  to  several 
causes.  In  the  first  place,  microscope  objectives  have  been  naturally 
constructed  to  give  the  clearest  image  to  the  eye ;  that  is,  the  visual 
image,  as  it  is  sometimes  called,  is  for  microscopic  observation  of 
prime  importance.  The  actinic  or  photographic  image,  on  the  other 
hand,  is  of  prime  importance  for  photography.  For  the  majority 
of  microscopic  objects  transmitted  light  (§  85)  must  be  used,  not 
reflected  light  as  in  ordinary  vision.  Finally,  from  the  shortness  of 
focus  and  the  smallness  of  the  lenses,  the  proper  illumination  of  the 
object  is  accomplished  with  some  difficulty,  and  the  fact  of  the  lack 
of  sharpness  over  the  whole  field  with  any  but  the  lower  powers 
have  combined  to  make  photo-micrography  less  successful  than  ordi- 
nary macro-photography.  So  tireless,  however.,  have  been  the  efforts 


CH.  VII]         PHOTOGRAPHING  WITH  THE  MICROSCOPE  215 

of  those  who  believed  in  the  ultimate  success  of  photo-micrography, 
that  now  the  ordinary  achromatic  objectives  with  panchromatic  or 
isochromatic  plates  and  a  color  screen  give  good  results,  while  the 
apochromatic  objectives  with  projection  oculars  give  excellent  results, 
even  in  hands  not  especially  skilled.  The  problem  of  illumination, 
has  also  been  solved  by  the  construction  of  achromatic  and  apo- 
chromatic condensers  and  by  the  electric  and  other  powerful  lights  now 
available.  There  still  remains  the  difficulty  of  transmitted  light  and 
of  so  preparing  the  object  that  structural  details  stand  out  with  suffi- 
cient clearness  to  make  a  picture  which  approaches  in  definiteness 
the  drawing  of  a  skilled  artist. 

The  writer  would  advise  all  who  wish  to  undertake  photo-microg- 
raphy seriously  to  study  samples  of  the  best  work  that  has  been 
produced.  Among  those  who  showed  the  possibilities  of  photo-micro- 
graphs was  Col.  Woodward  of  the  U.  S.  Army  Medical  Museum.  The 
photo-micrographs  made  by  him  and  exhibited  at  the  Centennial 
Celebration  at  Philadelphia  in  1876  serve  still  as  models,  and  no 
one  could  do  better  than  to  study  them  and  try  to  equal  them  in 
clearness  and  general  excellence.  According  to  the  writer's  observa- 
tion no  photo-micrographs  of  histologic  objects  have  ever  exceeded 
those  made  by  Woodward,  and  most  of  them  are  vastly  inferior.  It 
is  gratifying  to  state,  however,  that  at  the  present  time  many  original 
papers  are  partly  or  wholly  illustrated  by  photo-micrographs,  and  no 
country  has  produced  works  with  photo-micrographic  illustrations 
superior  to  those  in  "  Wilson's  Atlas  of  Fertilization  and  Karyo- 
kinesis"  and  "Starr's  Atlas  of  Nerve  Cells,"  issued  by  the  Columbia 
University  Press. 

Most  excellent  photo-micrographs  appear  at  frequent  intervals  in 
all  the  great  biological  journals.  These  should  be  studied  by  the 
young  photographer  ambitious  to  equal  and  then  to  excel  the  best. 

§  338a.  Considerable  confusion  exists  as  to  the  proper  nomenclature  of 
photography  with  the  microscope.  On  the  Continent  the  term  micro-photog- 
raphy (micro-photographie)  is  very  common,  while  in  English  photo-microg- 
raphy and  micro-photography  mean  different  things.  Thus:  A  photo-micrograph 
is  a  photograph  of  a  small  or  microscopic  object  usually  made  with  a  micro- 
scope and  of  sufficient  size  for  observation  with  the  unaided  eye;  while  a  micro- 
photograph  is  a  small  or  microscopic  photograph  of  an  object,  usually  a  large 
object,  like  a  man  or  woman,  and  is  designed  to  be  looked  at  with  a  microscope. 


2l6  PHOTOGRAPHING  WITH  THE   MICROSCOPE        [Cn.  VII 

Dr.  A.  C.  Mercer,  in  an  article  in  the  Proc.  Amer.  Micr.  Soc.,  1886,  p.  131, 
says  that  Mr.  George  Shadbolt  made  this  distinction.  See  the  Liverpool  and 
Manchester  Photographic  Journal  (now  British  Journal  of  Photography],  Aug. 
15,  1858,  p.  203;  also  Sutton's  Photographic  Notes,  Vol.  Ill,  1858,  pp.  205-208. 
On  p.  208  of  the  last,  Shadbolt's  word  "  Photo-micrography  "  appears.  Dr. 
Mercer  puts  the  case  very  neatly  as  follows:  "  A  Photo-Micrograph  is  a 
macroscopic  photograph  of  a  microscopic  object;  a  micro-photograph  is  a 
microscopic  photograph  of  a  macroscopic  object."  See  also  Medical  News,  Jan. 
27,  1894,  p.  108. 

In  a  most  interesting  paper  by  A.  C.  Mercer  on  "  The  Indebtedness  of 
Photography  to  Microscopy,"  Photographic  Times  Almanac,  1887,  it  is  shown 
that:  "  To  briefly  recapitulate,  photography  is  apparently  somewhat  indebted 
to  microscopy  for  the  first  fleeting  pictures  of  Wedgewood  and  Davy  [1802], 
the  first  methods  of  producing  permanent  paper  prints  [Reede,  1837-1839], 
the  first  offering  of  prints  for  sale,  the  first  plates  engraved  after  photographs 
for  the  purpose  of  book  illustration  [Donne  &  Foucault,  1845],  the  photo- 
graphic use  of  collodion  [Archer  &  Diamond,  1851],  and  finally,  wholly  in- 
debted for  the  origin  of  the  gelatino-bromide  process,  greatest  achievement 
of  them  all  [Dr.  R.  L.  Maddox,  1871].  See  further  for  the  history  of  Photo- 
micrography, Neuhauss,  also  Bousfield. 

§  339.  As  the  difficulties  of  photo-micrography  are  so  much  greater 
than  of  ordinary  photography,  the  advice  is  almost  universal  that  no 
one  should  try  to  learn  photography  and  photo-micrography  at  the 
same  time,  but  that  one  should  learn  the  processes  of  photography 
by  making  portraits,  landscapes,  copying  drawings,  etc.,  and  then  when 
the  principles  are  learned  one  can  take  up  the  more  difficult  subject 
of  photo-micrography  with  some  hope  of  success. 

The  advice  of  Sternberg  is  so  pertinent  and  judicious  that  it  is 
reproduced:  "Those  who  have  had  no  experience  in  making  photo- 
micrographs are  apt  to  expect  too  much  and  to  underestimate  the 
technical  difficulties.  Objects  which  under  the  microscope  give  a 
beautiful  picture  which  we  desire  to  reproduce  by  photography  may 
be  entirely  unsuited  for  the  purpose.  In  photographing  with  high 
powers  it  is  necessary  that  the  objects  to  be  photographed  be  in  a 
single  plane  and  not  crowded  together  and  overlying  each  other. 
For  this  reason  photographing  bacteria  in  sections  presents  special 
difficulties  and  satisfactory  results  can  only  be  obtained  when  the 
sections  are  extremely  thin  and  the  bacteria  well  stained.  Even 
with  the  best  preparations  of  this  kind  much  care*  must  be  taken  in 
selecting  a  field  for  photography.  It  must  be  remembered  that  the 
expert  microscopist,  in  examining  a  section  with  high  powers,  has 
his  finger  on  the  fine  adjustment  screw  and  focuses  up  and  down  to 


CH.  VII]         PHOTOGRAPHING  WITH  THE  MICROSCOPE  217 

bring  different  planes  into  view.  He  is  in  the  habit  of  fixing  his  atten- 
tion on  the  part  of  the  field  which  is  in  focus  and  discarding  the  rest. 
But  in  a  photograph  the  part  of  the  field  not  in  focus  appears  in  a 
prominent  way,  which  mars  the  beauty  of  the  picture." 

APPARATUS  FOR  PHOTO-MICROGRAPHY 

§  340.  Camera.  —  For  the  best  results  with  the  least  expenditure 
of  time  one  of  the  cameras  especially  designed  for  photo-micrography 
is  desirable,  but  is  not  by  any  means  indispensable  for  doing  good 
work.  An  ordinary  photographic  camera,  expecially  the  kind  known 
as  a  copying  camera,  will  enable  one  to  get  good  results,  but  the  trouble 
is  increased,  and  the  difficulties  are  so  great  at  best  that  one  would 
do  well  to  avoid  as  many  as  possible  and  have  as  good  an  outfit  as 
can  be  afforded  (fig.  129). 

The  first  thing  to  do  is  to  test  the  camera  for  the  coincidence  of 
the  plane  occupied  by  the  sensitive  plate  and  the  ground-glass  or 
focusing  screen.  Cameras  even  from  the  best  makers  are  not  always 
correctly  adjusted. 

For  the  method  of  procedure  see  above,  §  334. 

The  majority  of  photo-micrographs  do  not  exceed  8  centimeters 
in  diameter  and  are  made  on  plates  8xn,  10X13,  or  13X18  centi- 
meters (3^X4!  in.,  4X5  in.,  or  5x7  in.). 

For  pictures  larger  than  these  it  is  best  to  make  small,  very  sharp 
negatives  of  moderate  enlargement  and  then  print  these  at  any 
desired  size  by  means  of  projection  apparatus  (see  under  enlargements, 

§  359)- 

§  341.  Work  Room.  —  It  is  almost  self-evident  that  the  camera 
must  be  in  some  place  free  from  vibration.  A  basement  room  where 
the  camera  table  may  rest  directly  on  the  cement  floor  or  on  a  pier 
is  excellent.  Such  a  place  is  almost  necessary  for  the  best  work  with 
high  powers.  For  those  living  in  cities,  a  time  must  also  be  chosen 
when  there  are  no  heavy  vehicles  moving  in  the  streets.  For  less 
difficult  work  an  ordinary  room  in  a  quiet  part  of  the  house  or  labora- 
tory building  will  suffice.  It  helps  much  to  have  rubber  corks  in 
the  lower  ends  of  the  table  legs.  The  legs  may  also  be  made  to 
stand  on  four  thick  pads  of  rubber  or  of  thick  woolen  cloth. 


2l8 


PHOTOGRAPHING  WITH  THE  MICROSCOPE         [Cn.  VII 


Finally  the  camera  and  microscope  can  be  placed  on  a  board  plat- 
form and  that  put  into  a  shallow  box  nearly  filled  with  sawdust  or 
dry  sea  sand. 


FIG.  129.     VERTICAL  MICROSCOPE  AND  CAMERA  FOR  PHOTO-MICROGRAPHY. 
(About  -fa  natural  size). 

T  Low  table  50  cm.  high,  50  cm.  wide,  and  70  cm.  long  with  leveling  screws 
in  the  legs  (Is)  and  a  drawer  with  combination  lock  (d  d). 

M     Microscope. 

N  Nitrogen-filled  mazda  lamp  of  100  watts  in  lantern  for  artificial  day- 
light glass  (a);  (sc)  lampsocket  and  supply  cable. 

VC  Vertical  camera  supported  by  the  revolving  rod  (vgr)  which  is  gradu- 
ated in  centimeters  and  half  centimeters.  The  camera  may  be  turned  aside 
as  shown  by  the  dotted  lines. 

Base  The  heavy  iron  base  and  pillar  (p)  supporting  the  revolving  rod 
(vgr),  which  in  turn  supports  the  camera. 

cs  Clamping  screws  to  fix  the  two  ends  of  the  camera  in  any  desired 
position. 

mr  Magnification  rod.  This  serves  to  hold  the  extension  of  the  camera 
at  the  right  point  for  any  desired  magnification,  then  the  camera  as  a  whole 
moves  up  and  down  on  the  graduated  rod  (vgr). 

rs  Clamp  to  fix  the  camera  at  any  desired  extension  on  the  magnification 
rod  (mr). 

jg     Focusing  glass  (§  334). 

le     Light  excluder  (fig.  133-134)- 


CH.  VII]         PHOTOGRAPHING  WITH  THE  MICROSCOPE  219 

The  photo-engravers  have  overcome  vibrations  by  suspending 
their  cameras,  or  using  spring  coils  as  a  part  of  the  support.  In  case 
of  real  need  this  method  would  serve  the  photographer  with  the  micro- 
scope. The  whole  apparatus  must  be  suspended  or  supported  on 
springs,  so  that  any  vibration  will  be  equal  for  all  parts.  The  small 
table  and  vertical  camera  lend  themselves  to  either  suspension  or 
support  on  a  platform  with  spiral  springs,  or  the  microscope  and 
camera  can  stand  directly  on  the  platform  (fig.  130). 


FIG.  130.     PLATFORM  WITH  SPIRAL  SPRINGS  FOR  THE  CAMERA  TO  NULLIFY 
VIBRATIONS  IN  PHOTOGRAPHING. 

§  342.  Arrangement  and  position  of  the  camera  and  the  micro- 
scope. —  For  much  photo-micrography  a  vertical  camera  and  micro- 
scope are  to  be  preferred.  Excellent  arrangements  were  perfected 
long  ago,  especially  by  the  French.  ('See  Moitessier). 

Vertical  photo-micrograph ic  cameras  are  now  commonly  made, 
and  by  some  firms  only  vertical  cameras  are  produced.  They  are 
exceedingly  convenient,  and  do  not  require  so  great  a  disarrangement 
of  the  microscope  to  make  the  picture  as  do  the  horizontal  ones.  The 
variation  in  size  of  the  picture  in  this  case  is  mostly  obtained  by  the 
objective  and  the  projection  ocular  rather  than  by  length  of  bellows. 
It  must  not  be  forgotten,  however,  that  penetration  varies  inversely 
as  the  square  of  the  power,  and  only  inversely  as  the  numerical  aper- 
ture ;  consequently  there  is  a  real  advantage  in  using  a  lo\v  power  of 
great  aperture  and  a  long  bellows  rather  than  an  objective  of  higher 
power  with  a  short  bellows.  A  horizontal  camera  is  more  convenient 
for  use  with  the  electric  light  also  (fig.  131). 


220 


PHOTOGRAPHING  WITH  THE  MICROSCOPE         [Cn.  VII 


For  convenience  and  rapidity  of  work  a  microscope  with  mechani- 
cal stage  is  necessary;  and  for  sections  where  it  is  desirable  to  have 
the  image  in  some  regular  position  a  revolving  stage  to  the  micro- 
scope helps  greatly  in  orienting  the  image  on  the  plate. 

It  is  also  an  advantage  to  have  a  tube  of  large  diameter  so  that  the 
field  will  not  be  too  greatly  restricted  (fig.  in).  In  some  microscopes 
the  tube  is  removable  almost  to  the  nose-piece  to  avoid  interfering 


Condenser 


Radiant 


FIG.   131.     HORIZONTAL  PROJECTION  MICROSCOPE  FOR  DRAWING  AND  FOR 
PHOTO-MICROGRAPHY. 

(About  ij<y  natural  size.     From  Optic  Projection). 

The  wiring  is  for  an  arc  lamp  with  right-angled  carbons.  The  condenser 
has  three  lenses  and  a  water  cell  between  the  two  plano-convex  ones. 

P  L  Concave  parallelizing  lens  to  supply  the  substage  condenser  with 
parallel  beams.  (See  also  fig.  115). 

Ms  45°  mirror  in  dotted  lines  beyond  the  ocular  to  reflect  the  image 
down  upon  the  surface  of  the  table  for  drawing. 

with  the  size  of  the  image.  The  substage  condenser  should  be  mov- 
able on  a  rack  and  pinion.  The  microscope  should  have  a  flexible 
pillar  for  work  in  a  horizontal  position.  While  it  is  desirable  in  all 
cases  to  have  the  best  and  most  convenient  apparatus  that  is  made, 
it  is  not  by  any  means  necessary  for  the  production  of  excellent  work. 
A  simple  stand  with  flexible  pillar  and  good  fine  adjustment  will 
answer. 

§  343.  Objectives  and  oculars  for  photo-micrography.  —  The 
belief  is  almost  universal  that  the  apochromatic  objectives  are  most 


CH.  VII]        PHOTOGRAPHING  WITH  THE  MICROSCOPE 


221 


satisfactory  for  photography.  They  are  employed  for  this  purpose 
with  a  special  projection  ocular.  Two  low  powers  are  used  without 
any  ocular.  Some  of  the  best  work  that  has  ever  been  done,  however, 
was  done  with  achromatic  objectives  (work  of  Woodward  and  others). 
One  need  not  desist  from  undertaking  photo-micrography  if  he  has 
good  achromatic  objectives.  From  a  somewhat  extended  series  of 
experiments  with  the  objectives  of  many  makers  the  good  modern 
achromatic  objectives  were  found  to  give  excellent  results  when  used 
without  an  ocular.  Most  of  them  also  gave  good  results  with  pro- 


FIG.  132.     HOME-MADE  OPTICAL  BENCH. 
(About  TV  natural  size.     From  Optic  Projection). 

This  is  composed  of  a  baseboard  on  which  are  fastened  two  rods  (t  t  t  t  t) 
to  serve  as  a  track  along  which  the  different  apparatus  blocks  can  be  moved. 

The  shaded  part  (as)  is  covered  with  asbestos  paper  to  avoid  any  danger 
from  fire. 

//  The  flanges  holding  the  sockets  for  inserting  the  different  pieces  of 
apparatus.  (Compare  fig.  109-112,  131). 

jection  oculars.  It  must  be  said,  however,  that  the  best  results  were 
obtained  with  the  apochromatic  objectives  and  projection  oculars. 
The  compensation  oculars  also  give  good  results.  It  does  not  seem 
to  require  so  much  skill  to  get  good  results  with  apochromatics  as 
with  achromatic  objectives.  The  majority  of  photo-micrographers 
do  not  use  the  Huygenian  oculars  in  photography,  although  excellent 
results  have  been  obtained  with  them.  An  amplifier  is  sometimes 
used  in  place  of  an  ocular.  Considerable  experience  is  necessary  in 
getting  the  proper  mutual  position  of  objective  and  amplifier.  The 
introduction  of  oculars  especially  designed  for  projection  has  led  to 
the  discarding  of  ordinary  oculars  and  of  amplifiers.  Oculars  restrict 
.he  field  very  greatly;  hence  the  necessity  of  using  the  objective  alone 
for  large  specimens. 


222  PHOTOGRAPHING  WITH  THE  MICROSCOPE         [Cn.  VII 

§  344.  Difference  of  visual  and  actinic  foci.  —  Formerly  there 
was  much  difficulty  experienced  in  photo-micrographing  on  account 
of  the  difference  in  actinic  and  visual  foci.  Modern  objectives  are 
less  faulty  in  this  respect  and  the  apochromatics  are  practically  free 
from  it.  Since  the  introduction  of  orthochromatic  or  isochromatic 
plates,  and  in  many  cases  the  use  of  color  screens,  but  little  trouble 
has  arisen  from  differences  in  the  foci.  This  is  especially  true  when 
mono-chromatic  light  and  even  when  petroleum  light  is  used.  In 
case  an  objective  has  its  visual  and  actinic  foci  at  markedly  different 
levels  it  would  be  better  to  discard  it  for  photography  altogether,  for 
the  estimation  of  the  proper  position  of  the  sensitive  plate  after  focus- 
ing is  only  guesswork  and  the  result  is  mere  chance.  If  sharp  pictures 
cannot  be  obtained  with  an  objective  when  petroleum  light  and  ortho- 
chromatic  plates  are  used,  the  fault  may  not  rest  with  the  objective, 
but  with  the  plate  holder  and  focusing  screen.  They  should  be  very 
carefully  tested  to  see  if  there  is  coincidence  in  position  of  the  focusing 
screen  and  the  sensitive  film  as  described  in  §  334. 

LIGHTING  FOR  PHOTO-MICROGRAPHY 

§  345.  Light.  —  The  best  light  is  sunlight.  That  has  the  defect 
of  not  always  being  available,  and  of  differing  greatly  in  intensity 
from  hour  to  hour,  day  to  day,  and  season  to  season.  The  sun  does 
not  shine  in  the  evening  when  many  workers  find  the  only  opportunity 
for  work.  Following  the  sunlight  the  electric  light  is  the  most  intense 
of  the  available  lights. 

For  many  specimens  daylight  gives  altogether  the  best  results, 
and  as  natural  daylight  is  not  constantly  available  the  photo-microg- 
rapher  has  now  at  his  disposal  the  artificial  daylight  by  the  use  of  a 
nitrogen-filled  mazda  lamp  and  daylight  glass.  The  lantern  for  this 
shown  in  fig.  129  was  found  to  be  excellent  and  the  results  obtained 
by  its  use  in  photographing  with  powers  up  to  the  1.5  mm.  homogeneous 
immersion  were  excellent.  Of  course  any  light  filters  which  are 
adapted  to  natural  daylight  would  serve  perfectly  with  the  artificial 
daylight. 

For  all  preparations  needing  a  yellow  color  screen  for  daylight,  a 
petroleum  or  kerosene  lamp  gives  good  results  for  the  majority  of 


CH.  VII]         PHOTOGRAPHING  WITH  THE  MfCROSCOPE  223 

low  and  moderate  power  work.  And  even  for  2  mm.  homogeneous 
immersion  objectives,  the  time  of  exposure  is  not  excessive  for  many 
specimens  (40  seconds  to  3  minutes).  This  light  is  cheap  and  easily 
obtained. 

A  lamp  with  flat  wick  about  40  mm.  wide  has  been  found  most 
generally  serviceable.  For  large  objects  and  low  powers  the  flame 
may  be  made  large  and  the  face  turned  toward  the  mirror.  This 
will  light  a  large  field.  For  high  powers  the  edge  toward  the  mirror 
gives  an  intense  light.  The  ordinary  glass  chimney  answers  well, 
especially  where  a  shield  is  used  (fig.  58). 

In  managing  the  light  for  photography  with  the  microscope,  follow 
the  directions  given  in  Ch.  II,  and  under  drawing  in  Ch.  VI.  See 
below  for  the  use  of  color  screens. 

§  346.  Objects  suitable  for  photo-micrographs.  —  While  almost 
any  large  object  may  be  photographed  well  with  the  ordinary  camera 
and  photographic  objective,  only  a  small  part  of  the  objects  mounted 
for  microscopic  study  can  be  photo-micrographed  satisfactorily. 
Many  objects  that  can  be  clearly  seen  by  constant  focusing  with  the 
fine  adjustment  appear  almost  without  detail  on  the  screen  of  the 
photo-micrographic  camera  and  in  the  photo-micrograph. 

If  one  examines  a  series  of  photo-micrographs  the  chances  are  that 
the  greater  number  will  be  of  diatoms,  plant  sections,  or  preparations 
of  insects.  That  is,  they  are  of  objects  having  sharp  details  and 
definite  outlines,  so  that  contrast  and  definiteness  may  be  readily 
obtained.  Stained  microbes  also  furnish  favorable  objects  when 
mounted  as  cover-glass  preparations,  but  these  give  color  images  and 
require  a  color  screen. 

For  success  with  preparations  of  animal  tissue  they  must  approxi- 
mate as  nearly  as  possible  to  the  conditions  more  easily  obtained  with 
vegetable  preparations.  That  is,  they  must  be  made  so  thin  and  be 
so  prepared  that  the  cell  outlines  have  something  of  the  definiteness 
of  vegetable  tissue.  It  is  useless  to  expect  to  get  a  clear  photograph 
of  a  section  in  which  the  details  are  seen  with  difficulty  when  studying 
it  under  the  microscope  in  the  ordinary  way. 

Many  sections  which  are  unsatisfactory  as  wholes  may  neverthe- 
less have  parts  in  which  the  structural  details  show  with  satisfactory 


224  PHOTOGRAPHING  WITH  THE  MICROSCOPE        [Cn.  VII 

clearness.  In  such  a  case  the  part  of  the  section  showing  details 
satisfactorily  should  be  surrounded  by  a  delicate  ring  by  means  of  a 
marker  (fig.  59-61).  If  one's  preparations  have  been  carefully  studied 
and  the  special  points  in  them  thus  indicated,  they  will  be  found  far 
more  valuable  both  for  ordinary  demonstration  and  for  photography. 
The  amount  of  time  saved  by  marking  one's  specimens  can  hardly 
be  overestimated.  The  most  satisfactory  material  for  making  the 
rings  is  shellac  colored  with  lampblack. 

Formerly  many  histologic  preparations  could  not  be  satisfactorily 
photographed.  Now  with  improved  section  cutters,  better  staining 
and  mounting  methods,  and  with  the  color  screens  and  isochromatic 
and  panchromatic  plates  (§  380)  almost  any  preparation  which  shows 
the  elements 'clearly  when  looking  into  the  microscope  can  be  satis- 
factorily photographed.  Good  photographs  cannot,  however,  be 
obtained  from  poor  preparations. 

In  photo-micrography  do  not  forget  the  three  ways  in  which  details 
of  structure  may  be  brought  out  clearly: 

(1)  By  difference  of  refraction  of  the  object  and  the  mounting 
medium  (refraction  images,  §  137). 

(2)  By  differential  staining  (color  images,  §  139). 

(3)  By  means  of  dark-ground  illumination  (§  117). 

EXPERIMENTS  IN  PHOTO-MICROGRAPHY 

§  347.  The  following  experiments  are  introduced  to  show  practi- 
cally just  how  one  would  proceed  to  make  photo-micrographs  with 
various  powers,  and  be  reasonably  certain  of  fair  success.  If  one 
consults  prints  or  the  published  figures  made  directly  from  photo- 
micrographs it  will  be  seen  that,  excepting  diatoms  and  bacteria, 
the  magnification  ranges  mostly  between  10  and  150  diameters. 

§  348.  Focusing  in  photo-micrography.  —  For  rough  focusing  and 
as  a  guide  for  the  proper  arrangement  of  the  object  one  uses  a  ground- 
glass  screen,  as  in  gross  photography.  With  the  ground-glass  screen 
one  can  judge  of  the  brilliancy  and  evenness  of  the  illumination  more 
accurately  than  in  any  other  way.  For  final  and  exact  focusing  two 
principal  methods  are  employed: 

(a).     A  focusing  glass  is  used  either  with  a  clear  screen  or  in  a 


CH.  VII]         PHOTOGRAPHING  WITH  THE  MICROSCOPE  225 

board  screen,  as  described  above  (§  334).  The  latter  method  is  like 
focusing  with  the  compound  microscope  and  a  positive  ocular.  If 
the  focusing  glass  is  set  properly  the  focus  should  be  easily  and  accu- 
rately determined. 

In  whatever  way  one  focuses  for  photo-micrography  a  difficulty 
often  appears.  No  matter  how  perfect  the  focus  of  the  microscope 
the  picture  may  be  out  of  focus.  This  may  be  due  to  either  one  of  two 
things:  (i)  the  focusing  screen  or  focusing  glass  may  not  be  in  the 
right  position  to  make  the  image  sharp  on  the  sensitive  plate.  (2)  The 
microscope  may  get  out  of  focus  while  the  picture  is  being  made.  The 
reason  for  this  change  may  be  the  gradual  settling  down  of  the  tube 
of  the  microscope.  This  may  be  a  fault  of  the  fine  or  of  the  coarse 
adjustment.  It  is  a  good  plan  to  focus  the  object  carefully  and  after 
10  or  15  minutes  to  see  if  the  focus  is  still  good.  If  the  microscope 
will  not  stay  in  focus  one  cannot  get  a  good  picture.  In  that  case 
it  is  necessary  to  study  the  apparatus  and  see  which  part  of  the 
mechanism  is  at  fault. 

§  349.  Photo-micrographs  of  20  to  50  diameters.  —  For  pictures 
under  15  to  20  diameters  it  is  better  to  use  the  camera  for  embryos 
with  the  objective  in  the  end  of  the  camera,  and  the  special  micro- 
scope stand  for  focusing  (fig.  126). 

For  pictures  at  25  to  50  diameters  one  may  use  the  microscope  with 
a  low  objective,  20  to  35  mm.  equivalent  focus,  and  no  ocular  (fig.  in). 
The  object  is  placed  on  the  stage  of  the  microscope  and  focused  as 
in  ordinary  observation.  If  a  vertical  microscope  is  used  the  light 
from  the  petroleum  lamp  or  other  artificial  light  is  reflected  upward 
by  the  mirror.  It  may  take  some  time  to  get  the  whole  field  lighted 
evenly.  In  some  cases  it  may  be  advisable  to  discard  the  condenser 
and  use  the  mirror  only.  For  some  purposes  one  will  get  a  better 
light  by  placing  the  bull's  eye  or  other  condenser  between  the  lamp 
and  the  mirror  to  make  the  rays  parallel  or  even  to  make  a  sharp 
image  of  the  lamp  flame  on  the  mirror.  Remember  also  that  in  many 
cases  it  is  necessary  to  have  a  color  screen  between  the  source  of  light 
and  the  object  (§  366). 

For  a  horizontal  camera  it  is  frequently  better  to  swing  the  mirror 
entirely  out  of  the  way  and  allow  the  light  to  enter  the  condenser 


226 


PHOTOGRAPHING  WITH  THE  MICROSCOPE         [Cn.  VII 


directly  (fig.  131).     When  the  light  is  satisfactory,  as  seen  through 
an  ordinary  ocular,  remove  the  ocular. 

(a)  Photographing  without  an  ocular.  —  After   the  removal  of  the 
ocular  put  in  .the  end  of  the  tube  a  lining  of  black  velvet  to  avoid 

reflections.  Connect  the  microscope  with 
the  camera,  making  a  light  tight  joint, 
and  focus  the  image  on  the  focusing 
screen.  One  may  make  a  light-tight  con- 
nection by  the  use  of  black  velveteen  or 
more  conveniently  by  the  double  metal 
hood  which  slips  over  the  end  of  the  tube 
of  the. microscope,  and  into  which  fits  a 
metal  cylinder  on  the  lower  end  of  the 
camera  (fig.  133-134).  In  figure  134  the 
connection  has  been  made. 

It  will  be  necessary* to  focus  down 
considerably  to  make  the  image  clear. 
Lengthen  or  shorten  the  bellows  to  make 
the  image  of  the  desired  size,  then  focus 
with  the  utmost  care.  In  case  the  field 
is  too  much  restricted  on  account  of  the 
tube  of  the  microscope,  remove  the  draw- 
tube.  When  all  is  in  readiness  it  is  well 
to  wait  for  three  to  five  minutes  and 
then  to  see  if  the  image  is  still  sharply 
focused.  If  it  has  become  out  of  focus 
simply  by  standing,  a  sharp  picture  could 
not  be  obtained.  If  it  does  not  remain 
in  focus,  something  is  faulty.  When 
the  image  remains  sharp  after  focusing 
make  the  exposure.  From  20  to  60  sec- 
onds will  usually  be  sufficient  time  with  medium  plates  and  light  as 
described.  If  a  color  screen  is  used  it  will  require  50-300  seconds, 
i.  e.,  2  to  5  times  as  long,  for  a  proper  exposure  (§  372). 

(b)  Photographing  with   a    projection  ocular.  —  If   the   object  is 
small  enough  to  be  included  in  the  field  of  a  projection  ocular  (fig. 


FIG.  133.  LIGHT  EXCLUDER 
FOR  CONNECTING  THE  CAM- 
ERA AND  MICROSCOPE. 

(About  j  natural  size). 

1  The  front  board  of  the 
camera. 

2  Connecting  piece  to  fit 
over  i  and  extend  down  into  j. 

3  Piece  to  fit  over  the  up- 
per end  of  the  tube  of   the 
microscope  and  to  receive  the 
lower  end  of  2  (compare  fig. 
134  where  the  parts  are  to- 
gether as  in  making  an  expo- 
sure). 


CH.  VII]          PHOTOGRAPHING  WITH  THE  MICROSCOPE 


227 


137),  use  that  for  making  the  negative  as  follows:  Swing  the  camera 
around  so  that  it  will  leave  the  microscope  free  (fig.  129).  Use  an 
ordinary  ocular,  focus  and  light  the  object,  then  insert  a  projection 
ocular  in  place  of  the  ordinary  one,  and  swing  the  camera  back  over 
the  microscope.  It  is  not  necessary  to  use  an  ordinary  ocular  for  the 
first  focusing,  but  as  its  field  is  larger  it  is  easier  to  find  the  part  of 
be  photographed.  The  first  step  is  then 
to  focus  the  diaphragm  to  the  projection 
ocular  sharply  on  the  focusing  screen. 
Bring  the  camera  up  close  to  the  micro- 
scope and  then  screw  out  the  eye-lens 
of  the  ocular  a  short  distance.  Observe 
the  circle  of  light  on  the  focusing  screen 
to  see  if  its  edges  are  perfectly  sharp.  If 
not,  continue  to  screw  out  the  eye-lens 
until  it  is.  If  it  cannot  be  made  sharp 
by  screwing  it  out,  reverse  the  opera- 
tion. Unless  the  edge  of  the  light  circle, 
i.  e.,  the  diaphragm  of  the  ocular,  is 
sharp,  the  resulting  picture  will  not  be 
satisfactory. 

It  should  be  stated  that  for  the  X2 
projection  ocular  the  bellows  of  the  cam- 
era must  be  extended  about  30  or  40 
centimeters  or  the  diaphragm  cannot  be 

satisfactorily  focused  on  the  screen.  The  X4  projection  ocular  can 
be  focused  with  the  bellows  much  shorter.  For  either  projection 
ocular  the  screen  distance  can  be  extended  almost  indefinitely. 

When  the  diaphragm  is  sharply  focused  on  the  screen,  the  micro- 
scope is  focused,  that  is,  first  with  the  unaided  eye  then  with  the  fo- 
cusing glass.  The  exposure  is  made  in  the  same  way,  as  though  no 
ocular  were  used  (§  3490),  although  one  must  have  regard  to  the  greater 
magnification  produced  by  the  projection  ocular  and  increase  the  time 
accordingly;  thus  when  the  X4  ocular  is  used,  the  time  should  be  at 
least  doubled  over  that  when  no  ocular  is  employed.  The  time  will 
be  still  further  increased  if  a  color  screen  is  used  (§  376). 


Draw- 
Tube 

FIG.  134.     LIGHT  EXCLUDER 
FOR  PHOTO-MICROGRAPHY. 

(About  j  natural  size). 

In  this  figure  the  different 
parts  of  the  light  excluder  are 
in  position  for  making  an  ex- 
posure. 

1  The  front  board  of  the 
camera. 

2  The  intermediate  part 
connecting    the    camera    and 
the    hollow   cylinder  on    the 
upper  end  of  the  microscope 
draw-tube.       (Compare     fig. 


228  PHOTOGRAPHING  WITH  THE  MICROSCOPE         [Cn.  VII 

It  is  recommended  that  when  the  bellows  have  sufficient  length 
the  lower  projection  oculars  be  used,  but  with  short  bellows  the 
higher  ones. 

§  350.  Determination  of  the  magnification  of  the  photo-micro- 
graph. —  After  a  successful  negative  has  been  made,  it  is  desirable 
and  important  to  know  the  magnification.  This  is  easily  determined 
by  removing  the  object  and  putting  in  its  place  a  stage  micrometer. 
If  the  distance  between  two  or  more  of  the  lines  of  the  image  on  the 
focusing  screen  is  obtained  with  dividers  and  the  distance  measured 
on  one  of  the  steel  rules,  the  magnification  is  found  by  dividing  the 
size  of  the  image  by  the  known  size  of  the  object  (§  234).  If  now  the 
length  of  the  bellows  from  the  tube  of  the  microscope  is  noted,  say 
on  a  record  table  like  that  in  §  358,  one  can  get  a  close  approxi- 
mation to  the  power  at  some  other  time  by  using  the  same  optical 
combination  and  length  of  bellows. 

For  obtaining  the  magnification  at  which  negatives  are  made  it  is 
a  great  advantage  to  have  one  micrometer  in  half  millimeters  ruled 
with  coarse  lines  for  use  with  the  lower  powers,  and  one  in  o.i  and 
o.oi  millimeters  ruled  with  fine  lines  for  the  higher  powers  (fig.  80). 

§  351.  Photo-micrographs  at  a  magnification  of  100  to  150  diam- 
eters. —  For  this,  the  simple  arrangements  given  in  the  preceding 
section  will  answer,  but  the  objectives  must  be  of  shorter  focus,  8 
to  3  mm. 

§  352.  Lighting  for  photo-micrography  with  moderate  and  high 
powers.  (100  to  2500  diameters).  —  No  matter  how  good  one's 
apparatus,  successful  photo-micrographs  cannot  be  made  unless  the 
object  to  be  photographed  is  properly  illuminated.  The  beginner 
can  do  nothing  better  than  to  go  over  with  the  greatest  care  the 
directions  for  centering  the  condenser,  for  centering  the  source  of 
illumination,  and  the  discussion  of  the  proper  cone  of  light  and  lighting 
the  whole  field,  as  given  in  §  110-112.  Then  for  each  picture  the 
photographer  must  take  the  necessary  pains  to  light  the  object  prop- 
erly. An  achromatic  condenser  is  almost  a  necessity  (§  101).  Whether 
a  color  screen  should  be  used  depends  upon  judgment  and  that  can 
be  attained  only  by  experience.  In  the  beginning  one  may  try  with- 
out a  screen  and  with  different  screens  and  compare  results. 


CH.  VII] 


PHOTOGRAPHING  WITH  THE  MICROSCOPE 


229 


.Exc 

FIG.  135.  VIEW  OF  THE  BACK  LENS 
OF  THE  OBJECTIVE,  SHOWING  THE  CON- 
DENSER OUT  OF  CENTER  AND  CENTERED. 

Exc  The  spot  of  light  (Z>)  is  to  one 
side  of  the  center,  showing  that  the  optic 
axis  of  the  condenser  is  not  in  line  with 
that  of  the  microscope. 

C  The  spot  of  light  (D)  is  in  the  center, 
showing  that  the  optic  axis  of  the  con- 
denser and  microscope  are  in  line. 


A  plan  used  by  many  skilled  workers  is  to  light  the  object  and  the 
field  around  it  well  and  then  to  place  a  metal  diaphragm  of  the  proper 
size  in  the  camera  very  close 
to  the  plate  holder.  This  will 
insure  a  clean,  sharp  margin 
to  the  picture.  This  metal  dia- 
phragm must  be  removed  while 
focusing  the  diaphragm  of  the 
projection  ocular,  as  the  dia- 
phragm opening  is  smaller  than 
the  image  of  the  ocular  dia- 
phragm. 

If  the  young  photo-micfog- 
rapher  will  be  careful  to  select 
for  his  first  trials  objects  of 
which  really  good  photo-mi- 
crographs have  already  been 
made,  and  then  persists  with  each  one  until  fairly  good  results  are 
attained,  his  progress  will  be  far  more  rapid  than  as  if  poor  pictures 

of  many  different  things  were 
made.  He  should,  of  course, 
begin  with  low  magnifications. 
§  353.  Adjusting  the  objec- 
tive for  cover-glass.  —  After 
the  object  is  properly  lighted, 
the  objective,  if  adjustable, 
must  be  corrected  for  the 
thickness  of  cover.  If  one 
knows  the  exact  thickness  of 
the  cover  and  the  objective  is 
marked  for  different  thick- 
nesses, it  is  easy  to  get  the 
adjustment  approximately  cor- 
rect mechanically;  then  the 
final  corrections  depend  on  the  skill  and  judgment  of  the  worker. 
It  is  to  be  noted  too  that  if  the  objective  is  to  be  used  without  a 


Exc 

FIG.  136.  FIELD  OF  THE  MICROSCOPE 
SHOWING  THE  LlGHT  IN  THE  CENTER  AND 
TO  ONE  SIDE. 

C  Fl  The  light  is  in  the  center  and 
illuminates  the  object. 

Exc  Fl  The  light  is  at  one  side  of  the 
center  and  does  not  illuminate  the  object. 
(The  field  is  not  fully  lighted,  as  a  low 
power  is  used  to  center  the  object  and 
the  light). 


230 


PHOTOGRAPHING  WITH  THE  MICROSCOPE        [Cn.  VII 


FIG.  137.  PRO- 
JECTION OCULAR  IN 
SECTION. 

(About  \  natural 
Size). 


/     The  upper 
the  ( 


projection   ocular   the  tube-length   is  practically   extended   to   the 

focusing  screen,  and  as  the  effect  of  lengthening  the  tube  is  the  same 

as  thickening  the  cover-glass,  the  adjusting  collar  must  be  turned  to 
a  higher  number  than  the  actual  thickness  of  the 
cover  calls  for  (see  §  134). 

§  354.  Photographing  without  an  ocular.  - 
Proceed  exactly  as  described  for  the  lower  power, 
but  if  the  objective  is  adjustable  make  the  proper 
adjustment  for  the  increased  tube-length  (§  134). 
§  355.  Photographing  with  a  projection  ocular. 
-  Proceed  as  described  in  §  349  b,  only  in  this 
case  the  objective  is  not  to  be  adjusted  for  the 
extra  length  of  bellows.  .  If  it  is  corrected  for  the 
ordinary  ocular,  the  projection  ocular  then  pro- 
jects this  correct  image  upon  the  focusing-screen. 
§  356.  Photo-micrographs  at  a  magnification 
of  500  to  2000  diameters.  —  For  this  the  homo- 
geneous immersion  objective  is  employed,  and  as 
it  requires  a  long  bellows  to  get  the  higher  mag- 
nification with  the  objective  alone,  it  is  best  to 
use  the  projection  oculars. 

For  this  work  the  directions  given  in  §  104-107 
must  be  followed  with  great  exactness.  The  edge 
of  the  petroleum  lamp  flame  is  sufficient  to  fill  the 
field  in  most  cases.  With  many  objects  the  time 
required  with  good  lamplight  is  not  excessive; 
viz.,  40  seconds  to  3  minutes.  The  reason  of  this 
is  that  while  the  illumination  diminishes  directly 
as  the  square  of  the  magnification,  it  increases 
with  the  increase  in  the  numerical  aperture,  so 

that  the  illuminating  power  of  the  homogeneous  immersion  is  great 

in  spite  of  the  great  magnification. 

For  work  with  high  powers  a  stronger  light  than  the  petroleum 

lamp  is  employed  by  those  doing  considerable  photo-micrography. 

Good  wrork  may  be  done,  however,  with  the  petroleum  lamp. 
It  may  be  well  to  recall  the  statement  made  in  the  beginning,  that 


or 

eye  end  of  the  oc- 
ular; it  is  composed 
of  two  convex  and 
one  concave  lens 
and  serves  to  pro- 
ject the  real  image 
formed  by  the  ob- 
jective and  field  lens 
at  d  upon  the  screen 
or  photographic 
plate.  It  is  mov- 
able to  permit  of 
focusing  at  differ- 
ent screen  distances. 

2  Field  lens  of 
the  projection  oc- 
ular. 

d  Diaphragm 
where  the  real  im- 
age is  formed. 


CH.  VII]          PHOTOGRAPHING  WTH  THE  MICROSCOPE 


231 


the  specimen  to  be  photographed  must  be  of  special  excellence  for 
all  powers.  No  one  will  doubt  the  truth  of  the  statement  who  under- 
takes to  make  photo-micrographs  at  a  magnification  of  500  to  2000 
diameters. 

If  one  has  a  complete  outfit  with  electric  arc  light  the  time  required 
for  photographing  objects  is  much  reduced,  i.e.,  ranging  from  i  to 
20  seconds  even  with  the  color  screen.  As  the 
light  is  so  intense  with  the  arc  light  it  is  neces- 
sary to  soften  it  greatly  for  focusing.  Several 
thicknesses  of  ground-glass  placed  between  the 
lamp  and  the  microscope  will  answer.  These  are 
removed  before  taking  the  negative.  It  is  well 
also  to  have  a  water  bath  on  the  optical  bench 
to  absorb  the  radiant  heat.  This  should  be  in 
position  constantly  (see  fig.  in,  131). 

§  357.  Use  of  oculars  in  photo-micrography. 
-There  is  much  diversity  of  opinion  whether 
or  not  the  ordinary  oculars  used  for  observation 
should  be  used  in  photographing.  Excellent  re- 
sults have  been  obtained  with  them  and  also 
without  them. 

When  an  ocular  is  used  the  eye-lens  serves  to 
project  a  real  image  of  the  objective,  not  to  act 
as  a  magnifier  with  the  eye  as  an  ordinary  obser- 
vation ;    therefore  for  the  best  results  in  photog- 
raphy this  eye-lens    should   be   a  combination 
which  will  give  a  correct  image.     For  apochromatic  objectives  only 
the  projection  or  the  compensation  oculars  should  be  used,  not  or- 
dinary Huygenian  oculars.    The  projection  and  compensation  oculars 
work  well  with  the  best  high-angled  achromatic  objectives  also. 


FIG.  138.  ACH- 
ROMATIC SUBSTAGE 
CONDENSER  FOR 
PHOTO -MICROG - 

RAPHY. 

(From  Watson's 
Catalogue). 

1,2,3  The  three 
optical  parts  of  the 
condenser.  (Com- 
pare fig.  39  and  40, 
also  the  construc- 
tion of  objectives  in 
fig.  21  A  B  C  and 
note  that  the  con- 
denser is  like  an  in- 
verted objective.) 


232 


PHOTOGRAPHIC   ENLARGEMENTS 


[CH.  VII 


§  358.    Negative  record  in  photography. 

Name  No. 


Location 


Camera  

Date  

Exposure  

Objective 

Developer 

Ocular                                                .    . 

Fixer. 

Condenser         .                

Mag.  X  

Diaphragm  

Remarks     

Object  stained  with 

Color  screen 

• 

Plate 

Light  and  hour 

PROJECTION  APPARATUS  FOR  PHOTOGRAPHIC  ENLARGEMENTS 
§  359.  Enlarged  prints  of  small  negatives.  —  There  is  great 
advantage  in  making  pictures  of  large  objects  at  a  considerable 
distance  with  a  long-focus  objective,  so  that  the  perspective  will  be 
correct  and  all  levels  of  the  object  be  in  good  focus.  It  is  also  ad- 
vantageous to  make  pictures  of  microscopic  objects  without  undue 
enlargement;  then  there  is  greater  sharpness  of  the  object  as  a  whole. 
If  now  one  wishes  a  large  print,  any  good  negative  can  be  used 
and  a  print  obtained  of  almost  any  desired  enlargement  by  using  a 
photographic  objective  for  projecting  the  image  upon  the  photographic 
paper.  This  is  done  with  projection  apparatus  in  a  dark  room  as 
follows:  The  management  of  the  projection  apparatus  is  as  for  draw- 
ing. The  negative  is  placed  in  some  kind  of  a  holder  and  put  in  the 
cone  of  light  of  the  main  condenser  where  the  part  of  it  to  be  enlarged 
is  fully  illuminated.  An  erect  image  will  be  printed  on  the  paper 
if  the  film  side  of  the  negative  faces  the  paper  exactly  as  for  contact 
printing.  Of  course  if  it  is  desired  to  reverse  the  position  it  can  be 
done  by  turning  the  film  side  toward  the  source  of  light. 


CH.  VII]  PHOTOGRAPHIC  ENLARGEMENTS  233 

§  360.  Size  of  condenser  required.  —  The  general  law  is  that  the 
diameter  of  the  condenser  must  be  equal  to  or  somewhat  greater 
than  the  diagonal  of  the  negative  or  part  of  the  negative  to  be  enlarged. 
For  example  to  enlarge  the  whole  of  a  lantern  slide  negative  (85  x 
100  mm.),  the  condenser  should  have  a  diameter  of  14  cm.  For  a 
negative  100  X  125  mm.  the  condenser  should  be  18  cm.  in  diameter; 
for  one  125  x  175  mm.  the  condenser  should  be  23  cm.  in  diameter,, 
and  for  a  negative  200  X  250  mm.  the  condenser  should  be  35  cm.  in 
diameter. 

§  361.  Objectives  to  use  for  enlarging.  —  It  is  necessary  to 
use  an  objective  which  has  been  corrected  for  photography.  The 
ordinary  projection  objective  gives  a  good  visual  image,  but  not  a 
good  photographic  image.  The  iris  diaphragm  must  be  wide  open 
(§  289,  362). 

In  preparing  for  printing,  which  of  course  is  done  in  a  dark  room, 
put  some  white  paper  in  a  printing  frame  with  a  clear  glass  in  it. 
Hold  it  in  the  path  of  the  beam  from  the  projection  apparatus,  and 
either  by  moving  a  support  near  the  apparatus,  or  by  moving  the 
projection  apparatus,  get  the  desired  size  of  picture.  One  can  deter- 
mine the  exact  magnification  by  putting  a  lantern  slide  of  the  metric 
scale  (fig.  104)  in  place  of  the  negative  and  projecting  its  image 
upon  the  white  paper  in  the  printing  frame. 

§  362.  Focusing  and  printing.  —  Focus  the  image  of  the  negative 
as  sharply  as  possible.  Then  put  over  the  end  of  the  objective  a 
cover  of  some  kind  with  ruby  glass  in  it.  This  will  allow  the  light  to 
pass  in  part,  but  it  will  not  injure  the  photographic  paper  to  be  used. 

Place  in  the  printing  frame  some  developing  paper  like  cyco  or 
velox.  Place  the  printing  frame  in  position.  The  image  will  show 
clearly  on  the  paper  by  the  red  light.  When  the  frame  is  in  the  exact 
position  desired,  remove  the  cap  with  ruby  glass  and  make  the  ex- 
posure. With  an  arc  light  the  time  will  vary  from  about  2  to  10 
seconds,  depending  on  the  density  of  the  negative.  Cover  the  objec- 
tive, turn  off  the  arc  lamp,  and  develop  the  print  as  for  con  tact  printed 
pictures.  As  shown  in  §  289,  a  mazda  lamp  may  be  used  instead 
of  an  arc  light  for  enlarging.  If  the  rather  large  source  of  light 
in  the  no  volt  lamp  is  used,  a  diffuser  of  ground  glass  is  needed 


234  PHOTOGRAPHY  WITH  COLOR  SCREENS  [Cn.  VII 

to  avoid  the  shadows  between  the  filaments.  When  a  diffuser  is 
used  with  the  mazda  or  arc  light  the  diaphragm  of  the  objective 
can  be  closed  as  much  as  desired,  but  of  course  it  then  takes  a  much 
longer  exposure.  If  now  one  uses  a  6- volt  mazda  head-light  lamp 
by  inserting  a  transformer  in  the  circuit  for  the  alternating  cur- 
rent, or  by  using  a  storage  battery  for  the  direct  current,  the  fila- 
ment is  so  concentrated  that  the  source  may  be  treated  like  that 
of  an  arc  light,  and  no  diffuser  used.  This  makes  it  possible  to 
use  the  full  opening  of  the  objective.  The  candle  power  of  the 
6  volt  mazda  is  much  less  than  that  of  the  arc  light,  but  it  has 
the  advantage  of  requiring  no  attention  after  being  once  centered. 
§  363.  Printing  the  image  of  an  object  directly  on  the  paper.  - 
With  the  apparatus  set  up  exactly  as  for  drawing  or  for  printing 
enlargements,  one  can  expose  the  developing  photographic  paper  to 
the  sharply  focused  image  of  the  specimen.  Of  course  this  will  give 
a  negative  image,  all  the  lights  and  shades  being  reversed,  but  the 
outlines  and  proportions  are  perfect.  Such  pictures  serve  as  useful 
a  purpose  as  shade-correct  pictures  for  model  making  and  for  keeping 
a  record  of  one's  specimens. 

PHOTOGRAPHIC  REPRESENTATION  OF  VISUAL  APPEARANCES 
PANCHROMATIC  PHOTOGRAPHY  WITH  "COLOR  SCREENS 

§  364.     Five  methods  of  rendering  objects  visible.  — 

(1)  The  mounting  medium  and  the  object   must  have  different 
refractive  indices,  then  the  outline  of  the  object  or  of  its  details  are 
margined  by  dark  borders  (§  137,  refraction  images). 

(2)  The  object  or  its  details  must  have  a  different  color  from  the 
surrounding  medium  or  neighboring  objects  (color  images,  §  137). 

(3)  The  object  or  its  details  must  appear  self-luminous,  the  sur- 
rounding field  being    dark   (method    of    dark-ground  illumination, 
§  117).     For  large  objects,  an  illuminated  clock  face  on  a  dark  night 
is  a  good  illustration. 

(4)  If  reflected  light  is  used  some  parts  of  the  object  must  absorb 
the  light  and  some  parts  reflect  it;  the  different  parts  will  then  appear 
as  light  and  dark. 


CH.  VII]  PHOTOGRAPHY  WITH  COLOR  SCREENS  235 

(5)  If  transmitted  light  is  used  some  parts  of  the  object  must  be 
transparent  or  translucent  and  other  parts  opaque.  The  opaque  parts 
will  then  appear  dark  and  the  transparent  or  translucent  parts  light. 

Two,  four,  and  five  might  properly  be  called  absorption  images. 

§  365.  Photography  is  admirably  adapted  to  represent  the  visual 
appearances  of  both  naked  eye  and  of  microscopic  objects.  There 
is  only  one  difficulty  which  is  really  serious,  and  that  is  in  the  proper 
representation  in  black  and  white  of  the  various  colors. 

This  difficulty  is  inherent  in  the  sensitiveness  of  the  eye  to  colors 
and  the  unlike  sensitiveness  of  the  photographic  plate  to  the  same 
colors.  If  both  were  equally  and  similarly  sensitive,  then  the  photo- 
graphic representation  of  color  in  shades  or  tones  of  black  and  white 
would  have  the  same  brightness  as  the  different  colors  to  the  eye. 
But  the  eye  has  its  maximum  sensitiveness  in  the.  green  (fig.  139), 
while  the  photographic  plate  has  almost  all  of  its  sensitiveness  in 
the  violet-blue  end  of  the  spectrum.  Indeed  it  is  sensitive  to  a  part 
of  the  ultra  violet  which  is  wholly  dark  to  the  eye.  Hence  the  photo- 
graph represents  the  brilliant  red-orange-yellow-green  image  seen  by 
the  eye  as  dark,  while  the  relatively  dark  violet-blue  to  the  eye  is 
rendered  white  by  the  photographic  plate.  The  photographic  image 
of  colored  objects  is  then  a  kind  of  negative  of  the  same  image  to  the 
eye.  This  has  made  the  use  of  photography  unsatisfactory  where 
objects  have  color,  and  most  objects  in  nature  are  colored  more  or 
less;  and  one  of  the  greatest  triumphs  of  microscopic  science  has  been 
the  differentiation  of  details  of  structure  by  selective  staining. 

From  the  earliest  history  of  photography  the  inability  to  render 
the  colors  properly  or  in  actual  colors  has  been  greatly  deplored.  To 
give  the  proper  brightness  in  tones  of  black  and  white  to  colored 
objects  two  things  had  to  be  attained: 

(i)  The  photographic  plates  which  were  originally  sensitive  only 
to  the  violet-blue  end  of  the  spectrum  had  to  be  rendered  sensitive 
to  the  other  colors.  The  first  step  was  in  getting  plates  sensitive  to 
the  spectrum  as  far  as  the  yellow.  These  are  the  so-called  Isochro- 
matic  or  Orthochromatic  plates.  The  final  step,  was  to  get  plates 
sensitive  to  all  the  colors  of  the  spectrum,  including  the  orange  and 
red.  These  are  known  as  Panchromatic  or  Spectrum  plates. 


236 


PHOTOGRAPHY  WITH  COLOR  SCREENS 


[CH.  VII 


FIG.  139.     SENSITIVENESS  OF  THE  EYE  TO  THE  SPECTRUM  WITH  MODERATE 

ILLUMINATION. 

(Base  Line  =  Wave  lengths  x  250,000  times). 

As  shown  in  this  curve  the  normal  human  eye  with  moderate  illumination 
has  its  maximum  sensitiveness  at  about  wave  length  Xo.55/i,  that  is,  in  the 
green  next  the  yellow.  With  very  brilliant  light  the  greatest  sensitiveness  is 
in  the  yellow,  while  with  dim  light  it  moves  along  well  into  the  green.  (See 
§  406  for  designation  of  wave  lengths  in  microns,  etc.). 

Ultra-violet  Short  radiation  invisible  to  the  eye.  Compare  the  sensitive- 
ness of  the  photographic  plate  to  this  radiation  (fig.  140-142). 

Violet-blue     Radiation  at  the  blue  end  of  the  spectrum. 

Green     Radiation  in  the  middle  of  the  spectrum. 

Red     Radiation  at  the  red  end  of  the  spectrum. 

Infra-red     Long  radiation  invisible  to  the  eye. 

G  Y     Borderland  between  green  and  yellow. 

B  G     Borderland  between  blue  and  green. 

(2)  But  as  all  of  these  color-sensitive  plates  are  more  sensitive  to  the 
violet-blue  than  to  the  other  colors,  it  is  necessary  to  use  some  means 


Green 


I 
|GY| 


Red 


X0.7, 


X0.4/*  xo.5/i  XQ.6/1 

FIG.   140.     NORMAL  SPECTRUM  SHOWING  THE  SENSITIVENESS  OF  ^ORDINARY 
PHOTOGRAPHIC  PLATES. 

(After  Mees,  and  magnified  as  in  fig.  139). 

As  shown  in  this  curve,  the  ordinary  photographic  plate  is  sensitive  only 
in  the  blue  end  of  the  spectrum  including  the  ultra-violet,  the  maximum 
sensitiveness  being  at  about  wave  length  \o.4$[j..  It  is  insensitive  to  all  wave 
lengths  longer  than  about  Xo.52/i.  (Compare  with  fig.  139,  141-142). 


for  reducing  or  blocking  out  part  of  the  violet-blue  light  without 
interfering  with  the  action  of  the  other  colors  (§  367).     For  gaining 


CH.  VII] 


'HOTOGRAPHY  WITH  COLOR  SCREENS 


2.37 


X0.6u 

FIG.    141.      NORMAL  SPECTRUM   SHOWING    THE    SENSITIVENESS   OF   ORTHO- 
CHROMATIC   OR   ISOCHROMATIC   PLATES. 

(After  Mees;  magnification  as  in  fig.  139). 

These  plates  have  practically  the  same  sensitiveness  as  the  ordinary  plates 
except  that  the  sensitiveness  is  continued  through  the  green  and  yellow. 
(Compare  fig.  139,  140  and  142.) 

contrast  effects  it  was  necessary  to  devise  means  for  blocking  out 
special  parts  of  the  spectrum  (§  368).  These  selecting  media  are 
known  as  Color  Screens  or  Ray  Filters. 

COLOR  SCREENS  OR  RAY  FILTERS 

§  366.  Color  screens  or  ray  filters.  —  These  are  transparent, 
colored  bodies  which  select  the  wave  lengths  of  light  which  they 
transmit  and  absorb  the  other  waves,  or  they  diminish  more  or  less 
some  of  the  wave  lengths  and  transmit  the  others  with  very  slight 
loss.  The  color  of  such  a  screen  to  the  eye  will  be  determined  by  the 


xo.5/, 

FIG.  142.     NORMAL  SPECTRUM   SHOWING  THE  SENSITIVENESS  OF   PANCHRO- 
MATIC PLATES. 

(After  Mees;  magnification  as  in  fig.  139). 

Panchromatic  plates  have  the  maximum  sensitiveness  still  in  the  violet-blue, 
but  it  is  extended  to  include  the  red.     (Compare  fig.  139-141). 

light  which  it  transmits  in  the  greatest  quantity.     For  example,  if 
the  violet-blue  light    is    absorbed  the  remaining  light  will  appear 


238  PHOTOGRAPHY  WITH   COLOR   SCREENS  [Cn.  VII 

yellow,  while  if  green  and  red  are  absorbed  the  transmitted  light  will 
appear  blue;  if  violet-blue  and  green  are  absorbed  the  light  will  appear 
red,  and  if  violet-blue  and  red.  are  largely  absorbed  the  remaining 
light  will  appear  green. 

§  367.  Compensating  ray  filters.  —  These  are  filters  or  screens 
which  aid  the  panchromatic  photographic  plate  in  giving  a  black 
and  white  picture  of  colored  objects  which  shall  correspond  in  bright- 
ness to  the  different  colors  as  seen  by  the  eye. 

As  all  photographic  plates,  even  the  panchromatic  ones,  are  more 
sensitive  to  the  violet-blue  than  to  the  other  colors  of  the  spectrum 
(fig.  142),  the  effect  of  the  violet-blue  must  be  reduced,  hence  yellow 
screens  must  be  used  to  do  this  and  compensate  for  the  smaller  sen- 
sitiveness of  the  plate  for  the  other  parts  of  the  spectrum. 

Fortunately  the  great  photographic  manufacturers  have  made  a 
study  of  the  principles  of  color  screens  as  well  as  of  their  plates,  and 
they  supply  workers  with  data  showing  what  wave  lengths  of  light 
their  different  plates  are  sensitive  to,  and  the  wave  lengths  absorbed 
wholly  or  in  part  by  their  ray  filters.  They  also  give  advice  from 
abundant  experience  as  to  the  proper  combination  of  plate  and  color 
screen  to  get  the  best  effect  in  photographing  a  great  variety  of 
colored  objects.  By  using  this  information,  and  profiting  by  expe- 
rience, one  can  learn  to  photograph  almost  any  object  successfully. 

§  368.  Contrast  ray  filters.  —  These  are  filters  or  screens  by  the 
aid  of  which  strong  contrasts  in  black  and  white  are  given  to  various 
colored  objects  or  their  details.  As  given  in  the  general  statement  of 
the  basis  for  visibility  of  objects  and  their  details,  refraction  and 
opacity  are  of  prime  importance  for  securing  sharp  outlines.  Color 
images  are  also  of  the  greatest  advantage  in  differentiating  the  details 
of  microscopic  structure,  but  as  color  does  not  appear  in  the  ordinary 
photograph  the  differentiation  of  colored  objects  must  be  secured  by 
producing  shades  of  light  and  dark  up  to  complete  blackness  in  some 
cases.  For  example,  in  some  microscopic  specimens  important  details 
may  be  stained  violet  or  blue.  To  the  eye  these  violet  or  blue  objects 
stand  out  with  great  clearness.  In  the  photograph,  on  the  other  hand, 
without  special  help  from  a  color  screen,  they  are  wholly  lost  or  are 
so  faint  that  they  can  hardly  be  seen.  To  make  such  details  stand 


CH.  VII]  PHOTOGRAPHY  WITH  COLOR  SCREENS  239 

out  in  shades  of  black,  a  yellow  color  screen  absorbing  violet-blue 
and  allowing  the  other  colors  to  pass  is  used  with  a  plate  sensitive 
to  the  other  colors  to  be  photographed.  A  picture  is  thus  obtained 
which  shows  the  violet-blue  objects  in  black  and  the  other  details 
in  various  shades. 

A  contrast  color  screen  does  not  of  course  give  correct  brightness, 
but  the  purpose  in  using  it  is  to  bring  out  in  the  most  striking  manner 
the  form  of  certain  structures.  The  general  law  is:  For  contrast 
effects,  use  a  color  screen  which  absorbs  the  light  transmitted  normally 
by  the  colored  object,  but  allows  the  other  colors  to  pass. 

§  369.  Refraction  and  opacity  and  color  screens.  —  It  should 
not  be  forgotten  in  using  color  screens  and  color-sensitive  plates  that 
refraction  and  opacity  exert  their  full  effect  in  producing  the  final 
result.  The  color  screen  acts  only  to  suppress  or  lessen  certain  definite 
wave  lengths.  Refraction  and  opacity  tend  to  suppress  all  wave 
lengths  in  certain  limited  borders  or  definite  areas.  Hence  any 
stain  like  hematoxylin  which  tends  to  make  an  object  more  opaque 
to  all  parts  of  the  spectrum  will  increase  the  contrast  even  if  no  color 
screen  is  used. 

§  370.  Lessening  contrast. — With  some  specimens  it  is  necessary 
to  lessen  contrast  in  order  to  bring  out  details  of  structure.  One 
of  the  striking  examples  frequently  referred  to  is  whalebone.  A 
microscopic  section  of  this  has  a  reddish  appearance  by  transmitted 
light.  If  now  a  blue  screen  is  used  with  a  panchromatic  plate  the 
greatest  possible  contrast  is  obtained,  and  the  object  loses  all  detail 
in  the  photograph.  If  on  the  other  hand  a  red  screen  is  used  the 
photograph  shows  good  detail  and  the  general  appearance  is  like  that 
seen  by  the  eye  in  looking  into  the  microscope. 

The  general  law  is :  When  the  contrast  is  too  great  use  a  color  screen 
of  the  same  color  as  the  object,  and  of  course  a  plate  must  be  used 
sensitive  to  that  color. 

§  371.  Use  of  the  microspectroscope  in  photo-micrography.  —  If 
one  studies  his  specimens  with  the  microspectroscope  and  makes  sure 
exactly  what  light  is  transmitted  by  them,  it  will  be  possible  to  judge 
with  intelligence  what  plate  and  what  color  screen  to  use  to  bring 
out  in  the  most  satisfactory  manner  their  structural  appearances. 


240  PHOTPGRAPHY  WITH  COLOR  SCREENS  [Cn.  VII 

Fortunately  the  manufacturers  furnish  the  information  concerning 
their  plates  and  the  color  niters,  so  that  labor  is  spared  the  individual 
worker.  It  might  be  worth  while  for  him  to  check  up  the  color  screens 
occasionally  to  make  sure  that  they  have  not  deteriorated. 

§  372.  Time  of  exposure  for  photo-micrographs.  —  This  varies 
from  the  fraction  of  a  second  to  several  minutes,  depending  on  four 
factors: 

(1)  The  nature  and  intensity  of  the  light. 

(2)  The  magnification  of  the  microscope.     The  higher  the  mag- 
nification the  longer  must  be  the  exposure. 

(3)  The   transparency  of   the  specimen.     The  more   transparent 
the  shorter  the  exposures. 

(4)  The  thicker  or  deeper  the  color  of  the  ray  filter  the  longer  must 
be  the  exposure. 

LIGHT  FOR  PHOTO-MICROGRAPHY 

§  373.  Daylight.  —  This  has  served  for  some  of  the  best  photo- 
graphs which  have  ever  been  made.  If  it  is  not  available,  artificial 
daylight  obtained  by  using  daylight  glass  forms  a  very  good  substi- 
tute (§97). 

§  374.  Artificial  lights.  —  As  compared  with  daylight  all  ordinary 
forms  of  artificial  light  have  a  great  excess  in  the  red  end  of  the  spec- 
trum (see  fig.  36,  comparing  the  mazda  and  daylight).  This  excess 
in  the  red  end  has  the  advantage  that  it  partly  compensates  for  the 
excessive  sensitiveness  of  the  photographic  plate  for  violet-blue 
light.  For  many  objects  a  kerosene  lamp  is  excellent  for  photograph- 
ing by,  as  it  serves  for  both  light  and  color  screen. 

§  375.  Mutual  adaptation  of  color  screen  and  light.  —  As  the 
color  screen  is  for  a  very  definite  purpose  in  absorbing  certain  parts 
of  the  light  it  follows  that  the  character  of  the  light  and  that  of  the 
color  screen  must  be  mutually  adapted.  For  example  it  is  self-evi- 
dent that  the  same  color  screen  for  a  given  preparation  would  not  serve 
for  both  daylight  and  the  light  from  a  mazda  lamp  (see  fig.  36).  So 
also  the  same  color  screen  would  not  be  successful  if  used  both  for 
the  mazda  light  and  for  the  light  of  a  kerosene  flame. 


CH.  VII]       DEVELOPERS  AND  LIGHT  FOR  DEVELOPING  241 

For  the  most  successful  use  of  color  screens  and  different  light 
sources  one  should  have  curves  of  the  intensity  of  the  light  in  dif- 
ferent parts  of  the  visible  spectrum  like  that  for  the  mazda  lamp  and 
sunlight  (fig.  36).  Then  one  should  know  the  absorption  by  each 
color  filter  for  each  kind  of  light.  Knowing  these  facts  and  the 
absorbing  and  transmitting  qualities  of  his  specimens,  and  the  sensi- 
tiveness of  the  photographic  plates  used  one  could  make  intelligent 
selections  and  reasonably  expect  good  results. 

§  376.  Exposure  with  color  screens.  —  The  color  screen  naturally 
increases  the  time  of  exposure.  It  depends  on  the  color  and  density 
of  the  screen.  In  general  the  exposure  is  increased  from  2  to  5  times. 
The  increase  necessary  is  usually  given  by  the  manufacturers,  there- 
fore each  individual  worker  does  not  have  to  find  out  by  experiment. 
There  is  plenty  of  opportunity  for  the  use  of  his  judgment  with  the 
different  qualities  of  his  specimens  (see  also  §  372). 

§  377.  Developers.  —  It  is  best  to  use  the  developers  recom- 
mended by  the  manufacturers  of  the  plates  used.  The  experts 
employed  by  the  manufacturers  have  found  the  best  means  for  devel- 
oping the  plates,  and  it  is  safe  to  follow  their  advice.  One  usually 
has  a  choice  of  developers;  and  as  a  general  statement  it  should  be 
said  that  the  beginner  would  be  wise  to  prefer  a  slow  developer,  for 
it  allows  a  greater  latitude  than  a  rapid  developer.  In  general  a 
developer  containing  much  bromide  works  slowly  and  gives  very 
strong  contrasts.  Sometimes  this  is  desirable,  but  often  it  is  better 
to  get  the  soft  effects  that  come  with  a  small  amount  of  bromide.  If 
one  studies  the  little  manuals  sent  out  by  the  manufacturers  there 
will  be  found  formulae  which  give  the  various  effects  desired.  (See 
collateral  reading  suggested  at  the  end  of  the  chapter). 

§  378.  Light  to  develop  by.  —  The  light  which  can  be  used  in  the 
dark  room  depends  upon  the  sensitiveness  of  the  plates  or  the  printing 
paper  used.  The  more  sensitive  the  plates  or  paper  the  less  light. 
Furthermore  the  sensitiveness  to  the  different  wave  lengths  is  also 
important  to  consider.  If  the  plates  are  sensitive  only  to  the  violet- 
blue  of  the  spectrum,  the  dark  room  can  be  quite  brightly  lighted 
with  red  light  with  entire  safety.  If  isochromatic  or  orthochromatic 
plates  are  used  they  are  sensitive  to  the  spectrum  up  to  and  including 


242 


DEVELOPERS  AND   LIGHT   FOR  DEVELOPING       [Cn.  VII 


yellow 'and  hence  the  dark-room  light  must  exclude  those,  or  be  red 
only. 

For  panchromatic  plates  which  are  sensitive  to  all  wave  lengths 
the  only  safe  method  is  to  develop  in  total  darkness  for  any  light 
will  fog  the  plate  if  it  acts  sufficiently  upon  it.  Sometimes  very  dark 
green  is  used,  for  the  eye  is  most  sensitive  to  green  if  the  light  is  very 
dim,  although  for  bright  light  the  eye  is  most  sensitive  to  yellow. 
But  to  be  able  to  see  clearly  enough  to  determine  the  stage  of  devel- 
opment by  the  green  light  dim  enough  to  be  safe  one  must  be  in  the 


FIG.  143.     DARK  ROOM  FOR  PHOTOGRAPHY  AND  DRAWING  IN  A  LARGE  ROOM. 
(From  Optic  Projection). 

dark  room  for  half  an  hour  or  more.  The  total  darkness  method  is 
safest.  One  learns  rather  quickly  to  work  in  total  darkness,  and  the 
time  during  which  development  goes  on  can  be  determined  by  count- 
ing seconds  or  a  signal  clock  ringing  minutes  or  an  alarm  clock  which 
can  be  set  at  the  beginning  for  the  estimated  time  can  be  used.  Or 
finally,  one  can  develop  in  a  tray  which  is  covered  so  that  no  light  can 
reach  the  plate;  then  the  ordinary  dark-room  light  can  be  turned  on 
from  time  to  time  to  see  when  the  estimated  period  for  development 
has  been  reached. 

It  is  far  safer  to  use  too  little  light  for  developing  rather  than  too 
much.  For  ordinary  or  for  isochromatic  plates  only  a  brief  glance 
occasionally  is  all  that  is  needed.  If  one  holds  the  plate  in  the  dark- 
room light  during  the  whole  development  or  for  a  considerable  time 
there  is  almost  always  a  thin  veil  of  fog  which  lessens  the  crispness  of 
the  picture. 


CH.  VII]  TIME   DEVELOPMENT   IN   PHOTOGRAPHY  243 

The  wisdom  of  the  advice  to  develop  isochromatic  or  ordinary 
plates  with  as  small  an  exposure  to  the  dark-room  light  as  possible 
can  be  demonstrated  by  the  beginner  in  the  following  experiment 
which  he  is  advised  to  try. 

Put  an  isochromatic  or  orthochromatic  plate  in  the  plate  holder. 
Pull  out  the  dark  slide  till  one  or  two  centimeters  of  the  film  is  ex- 
posed, then  leave  this  for  half  a  minute,  close  to  the  developing-room 
light.  Pull  out  the  slide  another  centimeter  or  two  and  expose 
again  to  the  dark-room  light.  Continue  till  the  entire  plate  has  been 
exposed.  The  last  segment  will  have  an  exposure  of  half  a  minute, 
next  to  the  last  a  whole  minute,  and  so  on.  Now  develop  the  picture 
in  the  ordinary  way  and  the  chances  are  that  the  plate  will  show 
very  marked  light  effects,  and  the  different  segments  in  proportion 
to  the  time  they  were  exposed  to  the  dark-room  light. 

§  379.  Time  development.  —  Assuming  that  the  correct  plate 
and  color  screen  is  used,  careful  experiments  made  in  the  scientific 
laboratories  of  the  large  plate  manufacturers  have  shown  that  the 
best  method  of  developing  photographic  negatives  is  that  of  devel- 
oping a  definite  time  at  a  definite  temperature  of  the  developer.  The 
time  and  temperature  must  of  course  be  determined  for  the  special 
plate  and  composition  of  developer  to  be  used.  The  variable  then 
is  the  exposure  of  the  plate.  A  perfectly  timed  plate  will  contain 
all  the  desired  detail  in  the  shadows  and  just  sufficient  density  in 
the  high  lights  so  that  the  print  will  be  sufficiently  white.  The 
deepest  shadows  in  such  a  negative  will  be  almost  perfectly 
transparent. 

A  convenient  and  safe  method  of  developing  plates  by  the  time 
method  without  having  the  room  absolutely  dark  and  without  expos- 
ing the  plate  to  any  harmful  light,  is  the  following:  The  dark-room 
safelight  is  directed  away  from  the  developing  tray  and  a  shield  put 
in  position  to  further  screen  it.  An  alarm  or  other  large-faced  clock, 
with  second  hand,  is  put  close  to  the  safelight.  This  light  may  then  be 
very  dim  and  still  illuminate  the  clock  face  sufficiently.  If  using 
isochromatic  or  orthochromatic  plates  the  red  safelight  is  good, 
but  if  panchromatic  or  spectrum  plates  are  used  the  green  safelight 
is  better.  The  exceedingly  minute  amount  of  light  reaching  the 


244  PHOTOGRAPHS  IN  COLORS  [Cn.  VII 

plate  from  the  safelight  as  here  recommended  can  cause  no  damage 
(Henry  Phelps  Gage,  Optical  Department,  Corning  Glass  Works). 

§  380.  Choice  of  plates  and  color  screens.  —  The  hints  given 
in  the  little  manuals  sent  out  by  the  manufacturers  on  request  by 
their  patrons  give  excellent  hints  for  the  selection  of  plates  and  color 
screens  for  a  wide  variety  of  objects.  The  beginner  cannot  do  better 
than  to  follow  those  suggestions  faithfully,  until  his  own  experience 
enables  him  to  supplement  those  suggestions.  Finally,  of  course,  one 
wishes  to  be  able  to  use  his  own  judgment. 

In  general,  if  any  color  is  present  in  the  object  to  be  photographed 
one  will  have  better  success  with  isochromatic  or  orthochromatic 
plates,  which  are  sensitive  to  violet-blue,  green,  and  yellow,  than  with 
the  ordinary  plates,  which  are  only  sensitive  to  the  violet-blue  of  the 
spectrum  (fig.  140-141).  If  the  colors  involved  contain  orange  and 
red  the  isochromatic  plates  are  not  adequate,  and  one  must  then 
use  panchromatic  or  spectrum  plates,  sensitive  to  all  wave  lengths 
(fig.  142). 

For  the  color  screen  to  employ,  remember  that  color  screens  are 
not  of  real  use  for  ordinary  plates  sensitive  only  to  violet  and  blue. 
For  isochromatic  plates  yellow  color  screens  are  very  helpful  for 
reducing  the  excessive  effect  of  the  violet  and  blue  (§  367)  or  for  cut- 
ting them  out  altogether  in  getting  contrast  effects  (§  368).  The  same 
is  true  for  panchromatic  plates,  only  here  a  wider  range  of  color 
screens  can  be  used  to  get  any  desired  contrast  or  compensating  effect. 

COLOR  PHOTOGRAPHY 

§  381.  Photographs  in  natural  colors.  —  This  has  been  the  aim 
of  experts  in  photography  ever  since  its  first  invention.  Lately 
methods  have  been  devised  by  which  surprisingly  true  color  photo- 
graphs have  been  produced.  These  color  pictures  are  better  adapted 
to  large  objects  than  to  those  with  fine  details  such  as  are  observed 
with  the  microscope.  Still,  many  objects  are  fairly  well  represented 
in  photo- micrographs. 

The  author's  experience  in  color  photography  has  been  limited 
to  the  "Autochrome  Process"  (colored  starch  grain  process).  The 
directions  in  the  small  manual  sent  out  with  the  plates  are  very  clear, 


CH.  VII]  PHOTOGRAPHS   IN   COLORS  245 

and  any  one  familiar  with  the  ordinary  photographic  processes  can 
succeed  in  color  photography.  It  may  be  said  in  passing  that  the 
pictures  taken  by  this  process  are  transparencies  and  must  be  looked 
at  as  such  to  bring  out  the  colors.  Furthermore,  as  colors  are  truly 
rendered  only  in  daylight  or  by  artificial  daylight  these  transparencies 
must  be  illuminated  by  natural  or  artificial  daylight  for  a  true  render- 
ing of  the  color. 

While  these  pictures  cannot  be  used  as  negatives  to  give  paper 
prints  in  colors,  they  can  be  used  as  colored  pictures  to  get  the 
proper  negatives  for  printing  by  the  three-color  process,  so  that  with 
a  good  autochrome  transparency,  colored  pictures  for  books  and 
magazines  can  be  produced  without  any  hand  being  taken  in  the 
process  by  an  artist;  and  for  many  things  the  transparency  gives  a 
truth  and  delicacy  in  coloring  not  attainable  by  the  artist's  brush. 

COLLATERAL  READING  FOR  CHAPTER  VII 

Optic  Projection,  by  S.  H.  &  H.  P.  Gage. 

The  Wratten  Booklets  on  Photographic  Plates  and  Color  Filters. 
The  Photography  of  Colored  Objects,  by  C.  E.  Kenneth  Mees. 
Photo-micrography.     Published  by  the  Eastman  Kodak  Co. 
Seed  Plates,  formulae  and  directions.     Eastman  Kodak  Co. 
Furnished  by  the  G.  Cramer  Dry  Plate  Company: 

Cramer's  Manual  on  Negative  Making  and  Formulas. 
Isochromatic  Landscape  Photography. 
The  Photographing  of  Color  Contrasts. 
Dry  Plates  and  Filters  for  Trichromatic  Work, 
Photo-micrographic  and  Spectrographic  Color  Filters. 

These  brochures  are  naturally  very  recent  and  give  the  meat  of  the  infor- 
mation at  present  available  on  the  kind  of  photographic  plates  available  and 
the  proper  color  filters  to  use  with  them  to  produce  the  best  effects  with  dif- 
ferent colored  objects  in  gross  photography  and  in  photo-micrography. 

For  the  sensitiveness  of  the  human  eye  to  the  different  parts  of  the  spec- 
trum see:  Herbert  E.  Ives,  Philosophical  Magazine,  Vol.  XXIV,  6th  ser. 
Dec.  1912,  pp.  853-863;  P.  G.  Nutting,  Transactions  of  the  Illuminating 
Engineering  Society,  1914,  pp.  633-642. 


CHAPTER   VIII 
MICRO-SPECTROSCOPE  AND  POLARISCOPE 

§  390.     Apparatus  and  material  for  Chapter  VIII. 

1.  Compound  microscope.  chlorophyll,     some     colored     fruits, 

2.  Micro-spectroscope  (§  392,400).  etc.  (§  412-419). 

3.  Watch-glasses    and    shell    vials,  5.    Micropolarizer  (§  421). 
slides,  and  covers  (§  410,  419).                          6.    Selenite  plate  (§  431). 

4.  Various  substances  for  exami-  7.    Various   doubly   refracting   ob- 
nation    (as    blood    and    ammonium      jects,  crystals,  textile  fibers,  starch, 
sulphide,    permanganate    of    potash,       section  of  bone  (§  430). 

§  391.  Visible  and  invisible  radiation.  —  From  any  primary 
source  of  light  energy  like  the  sun,  the  electric  arc,  etc.,  not  only  is 
the  energy  which  to  the  eye  is  appreciated  as  light,  but  wave  lengths  of 
energy  both  longer  and  shorter  than  those  affecting  the  eye  are 
given  off.  As  shown  in  fig.  144  the  segment  of  the  energy  spectrum 
which  is  visible  to  the  eye  is  exceedingly  limited,  being  included 
between  about  Xo.4/i  and  Xo.7ju.  Under  special  illumination,  waves 
shorter  than  Xo.4ju  and  longer  than  Xo.;^  can  be  seen,  but  the  exten- 
sion into  the  infra-red  or  the  ultra-violet  is  very  slight,  and  not  used 
for  ordinary  visual  purposes. 

It  is  fortunate  for  optical  instruments  that  the  visible  spectrum  is 
so  limited.  Indeed,  if  the  visible  spectrum  were  even  more  limited 
it  would  be  easier  to  obtain  perfect  images,  for  the  aberrations  arising 
from  the  different  wave  lengths  would  be  so  much  the  less. 

The  spectroscope  has  for  its  object  the  giving  of  information  con- 
cerning the  visible  spectrum,  and  it  has  proved  of  very  great  help 
indeed.  It  should  not  be  forgotten,  however,  that  the  color  effects 
produced  by  the  spectroscope  are  not  the  only  ones  and  in  some  ways 
not  the  most  important.  What  it  really  does  is  to  divide  up  the 
wave  lengths  in  groups,  and  in  absorption  phenomena  the  important 
thing  is  that  some  wave  lengths  are  not  present  or  are  cut  out  by  the 
absorbing  medium  and  hence  there  are  present  dark  bands  in  the 

246 


CH.  VIII]  MICRO-SPECTROSCOPE  247 

spectrum    (absorption   bands).     These   absorption   bands   could   be 
.seen  and  their  significance  appreciated  by  a  person  wholly  color  blind 
—  and  there  is  occasionally  such  a  person. 

§  392.  A  micro-spectroscope,  spectroscopic  or  spectral  ocular,  is 
a  direct-vision  spectroscope  in  connection  with  a  microscope  ocular. 
It  consists  of  a  direct-vision  spectroscope  prism  of  the  Amici  pattern, 
and  of  considerable  dispersion,  placed  over  the  ocular  of  the  microscope. 


Ultra-Violet  Infra-Red 

FIG.  144.    NORMAL  SPECTRUM  SHOWING  VISIBLE  AND  INVISIBLE  RADIATION. 

(Magnified  20,000  times  vertically  and  50,000  times  horizontally). 
(From  Optic  Projection). 

As  shown  in  white,  the  useful  radiation  for  vision  lies  between  Xo.4ju  and 
Xo.yju.  Under  favorable  conditions  the  eye  can  see  shorter  and  longer  radia- 
tions. 

Ultra-violet     Radiation  having  waves  shorter  than  XO.^JJL;    (Black). 

Infra-red  Radiation  with  waves  longer  than  Xo.y/z.  Radiation  up  to  a 
wave  length  of  X2^i  is  here  shown  in  black. 

This  direct  vision  or  Amici  prism  consists  of  a  single  triangular 
prism  of  heavy  flint  glass  in  the  middle  and  one  of  crown  glass  on 
each  side,  the  edges  of  the  crown  glass  prisms  pointing  toward  the 
base  of  the  flint  glass  prism,  i.e.,  the  edges  of  the  crown  and  flint 
glass  prisms  point  in  opposite  directions.  The  flint  glass  prism  serves 
to  give  the  dispersion  or  separation  into  colors,  while  the  crown-glass 
prisms  serve  to  make  the  emergent  rays  approximately  parallel  with 
the  incident  rays,  so  that  one  looks  directly  into  the  prism  along 
the  axis  of  the  microscope. 


248  MICRO-SPECTROSCOPE  [Cn.  VIII 

The  Amici  prism  is  in  a  special  tube  which  is  hinged  to  the  ocular 
and  held  in  position  by  a  spring.  It  may  be  swung  free  of  the  ocular. 
In  connection  with  the  ocular  is  the  slit  mechanism  and  a  prism  for 
reflecting  horizontal  rays  vertically  for  the  purpose  of  obtaining  a 
comparison  spectrum  (§  404).  Finally,  near  the  top  is  a  lateral  tube 
with  mirror  for  the  purpose  of  projecting  an  Angstrom  scale  of  wave 
lengths  upon  the  spectrum  (§  405,  fig.  145,  148). 

In  accordance  with  the  above  statements  the  dispersion  or  separa- 
tion into  colors  is  given  by  the  flint-glass  prism  or  prisms  and  follow- 
ing the  general  law  that  the  waves  of  shortest  length,  blue,  etc.,  will  be 
bent  most,  the  colors  have  the  position  indicated  in  fig.  139-142,  146, 
149.  But  if  one  looks  into  the  direct  vision  spectroscope  or  holds  the 
eye  close  to  the  single  prism  (fig.  145),  the  colors  will  appear  reversed 
as  if  the  red  were  more  bent.  The  explanation  of  this  is  shown  in  fig. 
145,  2,  where  it  can  be  readily  seen  that  if  the  eye  is  placed  at  E, 
close  to  the  prism,  the  different  colored  rays  appear  in  the  direction 
from  which  they  reach  the  eye  and  consequently  are  crossed  in  being 
projected  into  the  field  of  vision  and  the  real  position  is  inverted. 
The  same  is  true  in  looking  into  the  micro-spectroscope.  The  actual 
position  of  the  different  colors  may  be  determined  by  placing  some 
ground-glass  or  some  of  the  lens-paper  near  the  prism  and  observing 
with  the  eye  at  the  distance  of  distinct  vision. 

§  392a.  The  author  wishes  to  acknowledge  the  aid  rendered  by  Professor 
E.  L.  Nichols  in  giving  the  explanation  olTered  in  §  392. 

VARIOUS   KIXDS   OF   SPECTRA 

By  a  spectrum  is  meant  the  colored  bands  appearing  when  the 
light  traverses  a  dispersing  prism  or  a  diffraction  grating,  or  is  affected 
in  any  way  to  separate  the  different  wave  lengths  of  light  into  groups. 
When  daylight  or  some  good  artificial  light  is  thus  dispersed  one  gets 
the  appearance  so  familiar  in  the  rainbow. 

§  393.  Continuous  spectrum.  —  In  case  a  good  artificial  light, 
as  the  electric  light,  is  used,  the  various  rainbow  or  spectral  colors 
merge  gradually  into  one  another  in  passing  from  end  to  end  of  the 
spectrum.  There  are  no  breaks  or  gaps. 


CH.  VIII] 


MICRO-SPECTROSCOPE 


249 


FIG.  145.     DIAGRAM  OF  A  DIRECT-VISION  MICRO-SPECTROSCOPE. 

1  The  spectroscope  is  shown  in  position  on  the  microscope,  the  tube  of 
the  microscope  being  much  shortened  to  save  space. 

Stage,  the  stage  of  the  microscope  on  which  is  a  watch  glass  with  sloping 
sides. 

Objective     The  objective  of  the  microscope. 

S  Sf  S"  Screws  for  clamping  the  apparatus  and  for  changing  the  position 
of  parts. 

Slit  The  slit  of  the  spectroscope  between  the  ocular  lenses  in  the  position 
of  the  ocular  diaphragm. 

Hinge     The  hinge  on  which  the  prism  can  be  turned  off  the  ocular. 

Amid  prism  The  direct-vision  prism  composed  of  a  middle  flint  and  two 
crown-glass  prisms. 

Red  Yellou'  Blue     Arrangement  of  the  colors  as  they  emerge  from  the  prism. 

Scale  tube  and  Mirror  The  mirror  to  throw  light  into  the  scale  tube  and 
project  an  image  of  the  Angstrom  scale  into  the  field. 

2  Prism  showing  that  with  the  eye  close  to  the  prism  the  colors  seem  re- 
versed from  the  position  actually  occupied. 

3  Comp.  prism     The  prism  introduced  under  the  slit  and  serving  to  reflect 
up  into  the  microscope  a  spectrum  for  comparison  with  that  extending  along 
the  axis  of  the  microscope  from  below.     C  L     Liquid  in  the  tube  whose  spec- 
trum is  to  be  compared  with  that  of  the  liquid  in  the  watch  glass  on  the  stage 
of  the  microscope. 

4  The  slit  mechanism  and  comparison  prism  (p). 

5  S'     Set  screws  for  changing  the  width  and  length  of  the  slit. 


2  50 


SPECTROSCOPE  AND  VARIOUS  SPECTRA         [Cn.  VIII 


§  394.  Line  spectrum.  —  If  a  gas  is  made  incandescent,  the  spec- 
trum it  produces  consists,  not  of  the  various  rainbow  colors,  but  of 
sharp,  narrow,  bright  lines,  the  color  depending  on  the  substance. 

All  the  rest  of  the  spectrum  is  dark.  These  line  spectra  are  very 
strikingly  shown  by  metallic  vapors  heated  to  incandescence,  e.g. 


FIG.  146-147. 


G  F  E  D  C    B         A 

A  NORMAL  AND  A  PRISMATIC  SPECTRUM  OF  DAYLIGHT. 


FIG.  146.  NORMAL  SPECTRUM  OF  DAYLIGHT  SHOWING  THE  SEGMENTS  OF  COLOR, 
V  B  G  Y  O  R,  AND  THE  DARK  LINES,  HGFEDCBA. 

In  the  normal  spectrum  produced  by  a  grating  the  dispersion  is  directly 
proportional  to  the  wave  length  of  the  light;  hence  the  red  is  a  broad  band  and 
the  violet-blue  narrow.  (Compare  the  prismatic  spectrum  where  the  red  is 
narrow  and  the  blue  broad.) 

o.4)U  0.7/4,  the  wave  lengths  between  which  the  radiation  is  visible 
(see  fig.  144). 

FIG.  147.     PRISMATIC  SPECTRUM  OF  DAYLIGHT. 

As  glass  does  not  disperse  the  different  wave  lengths  in  direct  proportion 
to  their  length,  the  width  of  the  bands  of  color  are  strikingly  unlike  those  in 
the  normal  spectrum,  the  blue- violet  being  wide  and  the  red  very  narrow. 

sodium.     These  spectra  are  usually  obtained  by  heating  some  salt 
of  the  substance  (see  §  405). 

§  395.  Absorption  spectrum.  —  By  this  is  meant  a  spectrum  in 
which  there  are  dark  lines  or  bands  in  the  spectrum.  The  most 
striking  and  interesting  of  the  absorption  spectra  is  the  Solar  Spec- 
trum, or  spectrum  of  sunlight.  If  this  is  examined  by  a  good  spectro- 
scope it  will  be  found  to  be  crossed  by  dark  lines,  the  appearance 
being  as  if  one  were  to  draw  pen  marks  across  a  continuous  spectrum 


CH.  VIII]          SPECTROSCOPE  AND  VARIOUS  SPECTRA  251 

at  various  levels,  sometimes  apparently  between  the  colors  and  some- 
times in  the  midst  of  a  color.  These  are  the  so-called  Fraunhbfer 
lines.  Some  of  the  principal  ones  have  been  lettered  with  Roman 
capitals,  A,  B,  C,  D,  E,  F,  G,  H,  commencing  at  the  red  end.  The 
meaning  of  these  lines  was  for  a  long  time  unknown,  but  it  is  now 
known  that  they  correspond  with  the  bright  lines  of  a  line  spectrum. 
For  example,  if  sodium  is  put  in  the  flame  of  a  spirit  or  Bunsen  lamp 
it  will  vaporize  and  become  luminous.  If  this  light  is  examined  there 
will  be  seen  one  or  two  bright  yellow  bands  corresponding  in  position 
with  D  of  the  solar  spectrum  (fig.  146,  148).  If  now  the  spirit-lamp 
flame,  colored  by  the  incandescent  sodium,  is  placed  in  the  path  of 
the  electric  light,  and  it  is  examined  as  before,  there  will  be  a  continu- 
ous spectrum,  except  for  dark  lines  in  place  of  the  bright  sodium 
lines.  That  is,  the  comparatively  cool  yellow  light  of  the-  spirit- 
lamps  cuts  off  or  absorbs  the  intensely  hot  yellow  light  of  the  electric 
light;  and  although  the  spirit  flame  sends  a  yellow  light  to  the  spec- 
troscope it  is  so  faint  in  comparison  with  the  electric  light  that  the 
sodium  lines  appear  dark.  It  is  believed  that  in  the  sun's  atmosphere 
there  are  incandescent  metal  vapors  (sodium,  iron,  etc.),  but  that  they 
are  so  cool  in  comparison  with  the  rays  of  their  wave  length  in  the 
sun  that  the  cooler  light  of  the  incandescent  metallic  vapors  absorbs 
the  light  of  corresponding  wave  length,  and  is,  like  the  spirit-lamp 
flame,  unable  to  make  up  the  loss,  and  therefore  the  dark  lines  are 
present. 

§  396.  Absorption  spectra  from  colored  substances.  —  While 
the  solar  spectrum  is  an  absorption  spectrum,  the  term  is  more  com- 
monly applied  to  the  spectra  obtained  with  light  which  has  passed 
through  or  has  been  reflected  from  colored  objects  which  are  not 
self-luminous. 

It  is  the  special  purpose  of  the  micro-spectroscope  to  investigate 
the  spectra  of  colored  objects  which  are  not  self-luminous,  i.e.,  blood 
and  other  liquids,  various  minerals,  as  monazite,  etc.  The  spectra 
obtained  by  examining  the  light  reflected  from  these  colored  bodies 
or  transmitted  through  them  possess,  like  the  solar  spectrum,  dark 
lines  or  bands,  but  the  bands  are  usually  much  wider  and  less  sharply 
defined.  Their  number  and  position  depend  on  the  substance  or  its 


252 


SPECTROSCOPE  AND  VARIOUS  SPECTRA         [Cn.  VIII 


constitution  (fig.  148),  and  their  width,  in  part,  upon  the  thickness 
of  the  body.  With  some  colored  bodies,  no  definite  bands  are  present. 
The  spectrum  is  simply  restricted  at  one  or  both  ends  and  various 
of  the  other  colors  are  considerably  lessened  in  intensity.  This  is 
true  of  many  colored  fruits. 


£oUr 

$(itttru 


FIG.  148.     SPECTRA  TO  SHOW  DIFFERENT  KINDS  OF  ABSORPTION  BANDS. 

Solar  Spectrum  The  spectrum  of  daylight  showing  the  dark,  fixed  lines 
(Fraunhofer  lines)  A  B  C  D  E  F  G,  and  the  wave  lengths  in  microns,  .70,  .60 
.50,  .40. 

Sodium  The  spectrum  of  incandescent  sodium.  With  this  spectroscope  it 
is  a  single  bright  yellow  band  (D)  at  about  XO.SQJU,  all  the  rest  of  the  spectrum 
being  dark. 

Perman.  potash  The  spectrum  of  a  solution  of  permanganate  of  potash  and 
has  five  absorption  bands,  two  being  especially  dark  and  sharply  outlined. 

Methaemoglobin  The  spectrum  of  methaemoglobin  with  several  absorption 
bands,  the  two  in  the  yellow-green  being  darkest.  The  blue  end  of  the  spec- 
trum is  also  greatly  shortened. 

These  spectra  have  the  blue  end  at  the  right  instead  of  at  the  left  (compare 
fig.  144,  146-147). 


§  397.  Angstrom  and  Stoke's  law  of  absorption  spectra.  —  The 
waves  of  light  absorbed  by  a  body  when  light  is  transmitted  through 
some  of  its  substance  are  precisely  the  waves  radiated  from  it  when 
it  becomes  self-luminous.  For  example,  a  piece  of  glass  that  is 
yellow  when  cool  gives  out  blue  light  when  it  is  hot  enough  to  be 
self-luminous.  Sodium  vapor  absorbs  two  bands  of  yellow  light 
(D  lines) ;  but  when  light  is  not  sent  through  it,  but  itself  is  luminous. 


CH.  VIII]  ARRANGING  THE  SPECTROSCOPE  253 

and  examined  as  a  source  of  light,  its  spectrum  gives  bright  sodium 
lines,  all  the  rest  of  the  spectrum  being  dark  (fig.  148). 

§  398.  Law  of  color.  —  The  light  reaching  the  eye  from  a  colored 
solid,  liquid,  or  gaseous  body  lighted  with  white  light  will  be  that  due 
to  white  light  less  the  light  waves  that  have  been  absorbed  by  the 
colored  body.  Or  in  other  words,  it  will  be  due  to  the  wave  lengths 
of  light  that  finally  reach  the  eye  from  the  object.  For  example, 
a  thin  layer  of  blood  under  the  microscope  will  appear  yellowish 
green,  but  .a  thick  layer  will  appear  pure  red.  If  now  these  two  layers 
are  examined  with  a  micro-spectroscope,  the  thin  layer  will  show  all 
colors,  but  the  red  end  will  be  slightly,  and  the  blue  end  considerably, 
restricted,  and  some  of  the  colors  will  appear  considerably  lessened 
in  intensity.  Finally,  there  may  appear  two  shadow-like  bands,  or, 
if  the  layer  is  thick  enough,  two  well-defined  dark  bands  in  the  green 

(§  413). 

If  the  thick  layer  is  examined  in  the  same  way,  the  spectrum  will 
show  only  red  with  a  little  orange  light,  all  the  rest  being  absorbed. 
Thus  the  spectroscope  shows  which  colors  remain,  in  part  or  wholly, 
and  it  is  the  mixture  of  this  remaining  or  unabsorbed  light  that  gives 
color  to  the  object. 

§  399.  Complementary  spectra.  —  While  it  is  believed  that 
Angstrom's  law  (§  398)  is  correct,  there  are  many  bodies  on  which  it 
cannot  be  tested,  as  they  change  in  chemical  or  molecular  constitu- 
tion before  reaching  a  sufficiently  high  temperature  to  become  lumi- 
nous. There  are  compounds,  however,  like  those  of  didymium, 
erbium,  and  terbium,  which  do  not  change  with  the  heat  necessary  to 
render  them  luminous,  and  with  them  the  incandescent  and  ab- 
sorption spectra  are  mutually  complementary,  the  one  presenting 
bright  lines  where  the  other  presents  dark  ones  (Daniell). 

ADJUSTING  THE  MICRO-SPECTROSCOPE 

§  400.  The  micro-spectroscope,  or  spectroscopic  ocular,  is  put  in 
the  place  of  the  ordinary  ocular  in  the  microscope,  and  clamped  to 
the  top  of  the  tube  by  means  of  a  side  screw  for  the  purpose. 

§  401.  Adjustment  of  the  slit.  —  In  place  of  the  ordinary  dia- 
phragm with  circular  opening/  the  spectral  ocular  has  a  diaphragm 


254  ARRANGING  THE  SPECTROSCOPE  [Cn.  VIII 

composed  of  two  movable  knife  edges  by  which  a  slit-like  opening  of 
greater  or  less  width  and  length  may  be  obtained  at  will  by  the  use 
of  screws  for  the  purpose.  To  adjust  the  slit,  depress  the  lever 
holding  the  prism-tube  in  position  over  the  ocular,  and  swing  the 
prism  aside.  One  can  then  look  into  the  ocular.  The  lateral  screw 
should  be  used,  and  the  knife  edges  approached  till  they  appear 
about  half  a  millimeter  apart.  If  now  the  Amici  prism  is  put  back 
in  place  and  the  microscope  well  lighted,  one  will  see  a  spectrum  by 
looking  into  the  upper  end  of  the  spectroscope.  If  the  slit  is  too  wide, 
the  colors  will  overlap  in  the  middle  of  the  spectrum  and  be  pure  only 
at  the  red  and  blue  ends ;  and  the  Fraunhof er  or  other  bands  in  the 
spectrum  will  be  faint  or  invisible.  Dust  on  the  edges  of  the  slit 
gives  the  appearance  of  longitudinal  streaks  on  the  spectrum. 

§  402.  Mutual  arrangement  of  slit  and  prism.  —  In  order  that  the 
spectrum  may  appear  as  if  made  up  of  colored  bands  going  directly 
across  the  long  axis  of  the  spectrum,  the  slit  must  be  parallel  with 
the  refracting  edge  of  the  prism.  If  the  slit  and  prism  are  not  thus 
mutually  arranged,  the  colored  bands  will  appear  oblique,  and  the 
whole  spectrum  may  be  greatly  narrowed.  If  the  colored  bands  are 
oblique  grasp  the  prism  tube  and  slowly  rotate  it  to  the  right  or*to  the 
left  until  the  various  colored  bands  extend  directly  across  the  spectrum. 

§  403.  Focusing  the  slit.  —  In  order  that  the  lines  or  bands  in 
the  spectrum  shall  be  sharply  defined,  the  eye-lens  of  the  ocular 
should  be  accurately  focused  on  the  slit.  The  eye-lens  is  movable, 
and  when  the  prism  is  swung  aside  it  is  very  easy  to  focus  the  slit 
as  one  focused  for  the  ocular  micrometer  (§  240).  If  one  now  uses 
daylight  there  will  be  seen  in  the  spectrum  the  dark  Fraunhofer  lines 
(fig.  146,  148). 

To  show  the  necessity  of  focusing  the  slit,  move  the  eye-lens  down 
or  up  as  far  as  possible,  and  the  Fraunhofer  lines  cannot  be  seen. 
While  looking  in  to  the  spectroscope  move  the  ocular  lens  up  or  down, 
and  when  it  is  focused  the  Fraunhofer  lines  will  reappear.  As  the 
different  colors  of  the  spectrum  have  different  wave  lengths,  it  is 
necessary  to  focus  the  slit  for  each  color  if  the  sharpest  possible 
pictures  are  desired. 

It  will  be  found  that  the  eye-lens  of  the  ocular  must  be  farther  from 


CH.  VIII]  ARRANGING  THE  SPECTROSCOPE  255 

the  slit  for  the  sharpest  focus  of  the  red  end  than  for  the  sharpest 
focus  of  the  lines  at  the  blue  end.  This  is  because  the  wave  length 
of  the  red  is  markedly  greater  than  for  blue  light  (fig.  144). 

Longitudinal  dark  lines  on  the  spectrum  may  be  due  to  irregularity 
of  the  slit  or  to  the  presence  of  dust.  They  are  most  troublesome 
with  a  very  narrow  slit. 

§  404.  Comparison  or  double  spectrum.  —  In  order  to  compare 
the  spectra  of  two  different  substances  it  is  desirable  to  be  able  to 
examine  their  spectra  side  by  side.  This  is  provided  for  in  the  better 
forms  of  micro-spectroscopes  by  a  prism  just  below  the  slit,  so  placed 
that  the  light  entering  it  from  a  mirror  at  the  side  of  the  drum  shall 
be  totally  reflected  in  a  vertical  direction,  and  thus  parallel  with  the 
rays  from  the  microscope.  The  two  spectra  will  be  side  by  side,  with 
a  narrow  dark  line  separating  them.  If  now  the  slit  is  well  focused 
and  daylight  be  sent  through  the  microscope  and  into  the  side  to  the 
reflecting  or  comparison  prism,  the  colored  bands  and  the  Fraunhofer 
dark  lines  will  appear  directly  continuous  across  the  two  spectra. 
The  prism  for  the  comparison  spectrum  is  movable  and  may  be  thrown 
entirely  out  of  the  field  if  desired.  When  it  is  to  be  used,  it  is  moved 
about  halfway  across  the  field  so  that  the  two  spectra  shall  have 
about  the  same  width. 

§  405.  Scale  of  wave  lengths.  —  In  the  Abbe  micro-spectroscope 
the  scale  is  in  a  separate  tube  near  the  top  of  the  prism  and  at  right 
angles  to  the  prism-tube.  A  special  mirror  serves  to  light  the  scale, 
which  is  projected  upon  the  spectrum  by  a  lens  in  the  scale-tube. 
This  scale  is  of  the  Angstrom  form,  and  the  wave  lengths  of  any  part 
of  the  spectrum  may  be  read  off  directly,  after  the  scale  is  once  set 
in  the  proper  position,  that  is,  when  it  is  set  so  that  any  given  wave 
length  on  the  scale  is  opposite  the  part  of  the  spectrum  known  by 
previous  investigation  to  have  that  particular  wave  length.  The 
point  most  often  selected  for  setting  the  scale  is  opposite  the  sodium 
line,  where  the  wave  length  is,  according  to  Angstrom,  0.5892^.  In 
adjusting  the  scale,  one  may  focus  very  sharply  the  dark  sodium  line 
of  the  solar  spectrum  and  set  the  scale  so  that  the  number  0.589  is 
opposite  the  sodium  or  D  line,  or  a  method  that  is  frequently  used 
and  serves  to  illustrate  §  394-395,  is  to  sprinkle  some  salt  of  sodium 


256  DESIGNATING  WAVE  LENGTHS  OF  LIGHT       [Cn.  VIII 

(carbonate  of  sodium  is  good)  in  a  Bunsen  or  alcohol  flame  and  to 
examine  this  flame.  If  this  is  done  in  a  darkened  place  with  a  spectro- 
scope, a  narrow  bright  band  will  be  seen  in  the  yellow  part  of  the 
spectrum.  If  now  ordinary  daylight  is  sent  through  the  comparison 
prism,  the  bright  line  of  the  sodium  will  be  seen  to  be  directly  con- 
tinuous with  the  dark  line  at  D  in  the  solar  spectrum  (fig.  148).  By 
reflecting  light  into  the  scale-tube  the  image  of  the  scale  will  appear 
on  the  spectrum,  and  by  a  screw  just  under  the  scale-tube,  but  within 
the  prism- tube,  the  proper  point  on  the  scale  (0.589^1)  can  be  brought 
opposite  the  sodium  band.  All  the  scale  will  then  give  the  wave 
lengths  directly.  Sometimes  the  scale  is  oblique  to  the  spectrum. 
This  may  be  remedied  by  turning  the  prism-tube  slightly  one  way  or 
the  other.  It  may  be  due  to  the  wrong  position  of  the  scale  itself. 
If  so,  grasp  the  milled  ring  at  the  distal  end  of  the  scale-tube  and, 
while  looking  into  the  spectroscope,  rotate  the  tube  until  the  lines 
of  the  scale  are  parallel  with  the  Fraunhofer  lines.  It  is  necessary 
in  adjusting  the  scale  to  be  sure  that  the  larger  number,  0.70,  is  at 
the  red  end  of  the  spectrum. 

The  numbers  on  the  scale  should  be  very  clearly  defined.  If 
they  do  not  so  appear,  the  scale-tube  must  be  focused  by  grasping 
the  outer  tube  of  the  scale-tube  and  moving  it  toward  or  from  the 
prism-tube  until  the  scale  is  distinct.  In  focusing  the  scale,  grasp 
the  outer  scale-tube  with  one  hand  and  the  prism-tube  with  the  other, 
and  push  or  pull  in  opposite  directions.  In  this  way  one  will  be  less 
liable  to  injure  the  spectroscope. 

§  406.  Designation  of  wave  length.  —  Wave  lengths  of  light  are 
designated  by  the  greek  letter  X  followed  by  the  number  indicating 
the  length  in  some  fraction  of  a  meter.  See  fig.  144  where  the  visible 
spectrum  is  indicated  as  lying  between  wave  lengths  (X)  0.7/1  and 
o.4/x.  In  this  book  the  micron  (/*)  is  taken  as  the  unit  as  with  other 
minute  measurements.  Other  units  are  also  employed,  especially 
smaller  ones  so  that  the  wave  lengths  will  appear  as  whole  numbers 
instead  of  decimal  fractions.  The  two  other  units  having  the  greatest 
sanction  are  the  Angstrom  unit  (A.U.)  and  the  millimicron  (mju). 
See  §  246.  Expressed  in  Angstrom  units  the  limits  of  the  visible 
spectrum  would  be:  \jooo  to  X4ooo  A.U.;  and  if  in  millimicrons 
the  limits  are:  —  X7oo  to  X4OO  m/z.  The  wave  length  of  sodium 


TH.  VITT]  USING  THF  MICRO-SPECTROSCOPK  257 

light  would  be  expressed  thus  by  the  three  units:  —  AD  5892  A.U.; 
589.2  mfJL-  0.5892^. 

§  407.  Lighting  for  the  micro-spectroscope.  —  For  opaque  objects 
a  strong  light  should  be  thrown  on  them  either  with  a  concave  mirror 
or  condensing  lens.  For  transparent  objects,  the  amount  of  the 
substance  and  the  depth  of  the  color  must  be  considered.  As  a 
general  rule  it  is  well  to  use  plenty  of  light,  as  that  from  a  substage 
condenser  with  a  large  opening  in  the  diaphragm  or  with  the  dia- 
phragm entirely  open.  For  very  small  objects  and  thin  layers  of 
liquids  it  may  be  better  to  use  less  light.  One  must  try  both  methods 
in  a  given  case,  and  learn  by  experience. 

The  direct  and  the  comparison  spectra  should  be  about  equally 
illuminated.  One  can  manage  this  by  putting  the  object  requiring 
the  greater  amount  of  illumination  on  the  stage  of  the  microscope. 
In  lighting  it  is  found  in  general  that  for  red  or  yellow  objects,  lamp- 
light gives  very  satisfactory  results.  For  the  examination  of  blood 
and  blood  crystals,  the  light  from  a  petroleum  lamp  is  excellent.  For 
objects  with  much  blue  or  violet,  daylight  or  artificial  daylight  is 
best  (§  92). 

Furthermore,  one  should  be  on  his  guard  against  confusing  the 
ordinary  absorption  bands  with  the  Fraunhofer  lines  when  daylight 
is  used.  With  lamplight  the  Fraunhofer  lines  are  absent  and,  there- 
fore, not  a  source  of  possible  confusion. 

§  408.  Objective  to  use  with  the  micro-spectroscope.  —  If  the 
material  is  of  considerable  bulk,  a  low  objective  (16  to  50  mm.)  is 
to  be  preferred.  This  depends  on  the  nature  of  the  object  under 
examination,  however.  In  case  of  individual  crystals  one  should 
use  sufficient  magnification  to  make  the  real  image  of  the  crystal 
entirely  fill  the  width  of  the  slit.  The  length  of  the  slit  may  then  be 
regulated  by  the  screw  on  the  side  of  the  drum,  and  also  by  the  com- 
parison prism.  If  the  object  does  not  fill  the  whole  slit  the  white 
light  entering  the  spectroscope  with  the  light  from  the  object  might 
obscure  the  absorption  bands. 

In  using  high  objectives  with  the  micro-spectroscope  one  must 
very  carefully  regulate  the  light  (Ch.  II)  and  sometimes  shade  the 
object. 


258  USING  THE  MICRO-SPECTROSCOPE  [Cn.  VIII 

§  409.  Focusing  the  objective.  —  For  focusing  the  objective  the 
prism-tube  is  swung  aside,  and  then  the  slit  made  wide  by  turning  the 
adjustable  screw  at  the  "side.  If  the  slit  is  open  one  can  see  objects 
when  the  microscope  is  focused  as  with  an  ordinary  ocular.  After 
an  object  is  focused,  it  may  be  put  exactly  in  position  to  fill  the  slit 
of  the  spectroscope,  then  the  knife  edges  are  brought  together  till 
the  slit  is  of  the  right  width;  if  the  slit  is  then  too  long  it  may  be 
shortened  by  using  one  of  the  mechanism  screws  on  the  side,  or  if 
that  is  not  sufficient,  by  bringing  the  comparison  prism  farther  over 
the  field.  If  one  now  replaces  the  Amici  prism  and  looks  into  the 
microscope,  the  spectrum  is  liable  to  have  longitudinal  shimmering 
lines.  To  get  rid  of  these  focus  up  or  down  a  little  so  that  the 
microscope  will  be  slightly  out  of  focus. 

§  410.  Amount  of  material  necessary  for  absorption  spectra  and 
its  proper  manipulation.  —  The  amount  of  material  necessary  to 
give  an  absorption  spectrum  varies  greatly  with  different  substances, 
and  can  be  determined  only  by  trial.  If  a  transparent  solid  is  under 
investigation  it  is  well  to  have  it  in  the  form  of  a  wedge,  then  succes- 
sive thicknesses  can  be  brought  under  the  microscope.  If  a  liquid 
substance  is  being  examined,  a  watch  glass  with  sloping  sides  forms 
an  excellent  vessel  to  contain  it,  then  successive  thicknesses  of  the 
liquid  can  be  brought  into  the  field,  as  with  the  wedge-shaped  solid. 
Frequently  only  a  very  weak  solution  is  obtainable;  in  this  case  it 
can  be  placed  in  a  homoeopathic  vial,  or  in  some  glass  tubing  sealed 
at  the  end,  then  one  can  look  lengthwise  through  the  liquid  and  get 
the  effect  of  a  more  concentrated  solution.  For  minute  bodies  like 
crystals  or  blood  corpuscles,  one  may  proceed  as  described  in  the 
previous  section.  See  also  §  420. 

MICRO-SPECTROSCOPE  EXPERIMENTS 

§  411.  Put  the  micro-spectroscope  in  position,  arrange  the  slit 
and  the  Amici  prism  so  that  the  spectrum  will  show  the  various 
spectral  colors  going  directly  across  it  (§  402),  and  focus  the  slit.  This 
may  be  done  either  by  swinging  the  prism-tube  aside  and  proceeding 
as  for  the  ocular  micrometer  (§  240),  or  by  moving  the  eye  lens  of 
the  ocular  up  and  down  while  looking  into  the  micro-spectroscope  until 


CH.  VIII]  USING  THE  MICRO-SPECTROSCOPE  259 

the  dark  lines  of  the  solar  spectrum  are  distinct.  If  they  cannot 
be  made  distinct  by  focusing  the  slit,  then  the  light  is  too  feeble  or 
the  slit  is  too  wide.  With  the  lever  move  the  comparison  prism 
across  half  the  field  so  that  the  two  spectra  shall  be  of  equal  width. 
For  lighting,  see  §  407. 

§  411a.  If  cyie  does  not  possess  a  micro-spectroscope,  quite  satisfactory 
results  may  be  obtained  by  using  a  microscope  with  a  16  to  12  mm.  objective 
and  a  pocket  direct-vision  spectroscope  in  place  of  the  eye-piece.  (Bleile, 
Trans.  Amer.  Micr.  Soc.,  1900,  p.  8.) 

§  412.  Absorption  spectrum  of  permanganate  of  potash.  —  Make 
a  solution  of  permanganate  of  potash  by  putting  a  few  crystals  in  a 
watch  glass  of  water.  The  solution  should  be  of  a  strength  that  a 
stratum  of  3  to  4  mm.  thickness  will  be  transparent.  Place  the  watch 
glass  under  the  microscope.  Use  a  16  mm.  or  lower  objective  and 
open  widely  the  condenser  diaphragm;  light  strongly.  Look  into 
the  spectroscope  and  slowly  move  the  watch  glass  into  the  field.  Note 
carefully  the  appearance  with  the  thin  stratum  of  liquid  at  the  edge 
and  then  as  it  gradually  thickens  on  moving  the  watch  glass  still 
farther  along.  Count  the  absorption  bands  and  note  particularly 
the  red  and  blue  ends.  Compare  with  the  comparison  spectrum 
(fig.  148).  For  strength  of  solution  see  §  410. 

§  413.  Absorption  spectrum  of  blood.  —  Obtain  blood  from  a 
recently  killed  animal,  or  flame  a  needle,  and  after  it  is  cool  prick 
the  finger  two  or  three  times  in  a  small  area;  then  wind  a  handker- 
chief or  a  rubber  tube  around  the  base  of  the  finger  and  squeeze  the 
finger  with  the  other  hand.  Some  blood  will  ooze  out  of  the  pricks. 
Rinse  this  off  into  a  watch  glass  partly  filled  with  water.  Continue 
to  add  the  blood  until  the  water  is  quite  red.  Place  the  watch  glass 
of  diluted  blood  under  the  microscope  in  place  of  the  permanganate, 
using  the  same  objective,  etc.  Note  carefully  the  spectrum.  It 
would  be  advantageous  to  determine  the  wave  length  opposite  the 
center  of  the  dark  bands.  This  may  easily  be  done  by  setting  the 
scale  properly,  as  described  in  §  405.  Make  another  preparation, 
but  use  a  homoeopathic  vial  instead  of  a  watch  glass.  Cork  the  vial 
and  lay  it  down  upon  the  stage  of  the  microscope.  Observe  the 
spectrum.  It  will  be  like  that  in  the  watch-glass.  Remove  the  cork 


260 


USING  THE   MICRO-SPECTROSCOPE 


[Cn.  VIII 


and  look  through  the  whole  length  of  the  vial.  The  bands  will  be 
much  darker,  and  if  the  solution  is  thick  enough  only  red  and  a  little 
orange  will  appear.  Re-insert  the  cork  and  incline  the  vial  so  that 
the  light  traverses  a  very  thin  layer,  then  gradually  elevate  the  vial 
and  the  effect  of.  a  thicker  and  thicker  layer  may  be  seen.  Note 
especially  that  the  two  characteristic  bands  unite  and  form  one  wide 
band  as  the  stratum  of  liquid  thickens.  Compare  with  the  following: 
Add  to  the  vial  of  diluted  blood  a  drop  or  two  of  ammonium  sul- 
phide, such  as  is  used  for  a  reducing  agent  in  chemical  laboratories* 


so    so 


FIG.  149.     ABSORPTION  SPECTRUM  OF  ARTERIAL  AND  OF  VENOUS  BLOOD. 
(From  Gamgee  and  McMunn). 

1  Absorption  of  arterial  blood,  oxy-hemoglobin.     There  are  two  definite 
bands  between  wave  lengths  0.60/1  and  o.soju,  that  is,  in  the  yellow-green, 
and  the  blue  end  of  the  spectrum  is  cut  down  markedly. 

2  Single  dark  band   of  venous   blood,  hemoglobin,  in   the   yellow-green. 
The  blue  end  of  the  spectrum  is  less  cut  off  than  with  arterial  blood. 

A  B  C  D  E  F  G  H  Fixed  lines  of  the  solar  spectrum  .90,  .80,  .70,  .60,  .50,  .40; 
wave  lengths  in  microns  in  the  different  regions.  These  spectra  have  the  red 
end  at  the  left  instead  of  to  the  right,  as  is  now  more  usual  (fig.  144-147). 

Shake  the  bottle  gently  and  then  allow  it  to  stand  for  ten  or  fifteen 
minutes.  Examine  it  and  the  two  bands  will  have  been  replaced  by 
a  single,  less  clearly  defined  band  in  about  the  same  position.  The 
blood  will  also  appear  somewhat  purple.  Remove  the  cork  to 
admit  fresh  air,  then  shake  the  vial  vigorously,  and  the  color  will 
change  to  the  bright  red  of  fresh  blood.  Examine  it  again  with 
the  spectroscope  and  the  two  bands  will  be  visible.  After  five  or 
ten  minutes  another  examination  will  show  but  a  single  band.  In- 
cline the  bottle  so  that  a  thin  stratum  may  be  examined.  Note  that 
the  stratum  of  liquid  must  be  considerably  thicker  to  show  the  absorp- 
tion band  than  was  necessary  to  show  the  two  bands  in  the  first 


CH.  VIII]  USING  THE   MICRO-SPECTROSCOPE  261 

experiment.  Furthermore,  while  the  single  band  may  be  made  quite 
black  on  thickening  the  stratum,  it  will  not  separate  into  two  bands 
with  a  thinner  stratum.  In  this  experiment  it  is  very  instructive 
to  have  the  watch  glass  of  arterial  blood  under  the  microscope  and 
the  vial  of  blood  to  which  has  been  added  the  ammonium  sulphide 
in  position  for  a  comparison  spectrum. 

The  two-banded  spectrum  is  that  of  oxy-hemoglobin,  or  arterial 
blood;  the  single-banded  spectrum  of  hemoglobin  (sometimes  called 
reduced  hemoglobin)  or  venous  blood,  that  is,  the  respiratory  oxygen 
is  present  in  the  two-banded  spectrum  but  absent  from  the  single- 
banded  spectrum.  When  the  bottle  was  shaken  the  hemoglobin 
took  up  oxygen  from  the  air  and  became  oxy-hemoglobin,  as  occurs  in 
the  lungs,  but  soon  the  ammonium  sulphide  took  away  the  respiratory 
oxygen,  thus  reducing  the  oxy-hemoglobin  to  hemoglobin.  This 
may  be  repeated  many  times  (fig.  149). 

§  414.  Met-hemoglobin.  —  The  absorption  spectrum  of  met- 
hemoglobin  is  characterized  by  a  considerable  darkening  of  the  blue 
end  of  the  spectrum  and  of  four  absorption  bands,  one  in  the  red 
near  the  line  C  and  two  between  D  and  E,  nearly  in  the  place  of  the 
two  bands  of  oxy-hemoglobin;  finally  there  is  a  somewhat  faint, 
wide  band  near  F.  Such  a  met-hemoglobin  spectrum  is  best  obtained 
by  making  the  solution  of  blood  in  water  of  such  a  concentration  that 
the  two  oxy-hemoglobin  bands  run  together,  and  then  adding  three 
or  four  drops  of  a  0.1%  aqueous  solution  of  permanganate  of  potash. 
Soon  the  bright  red  will  change  to  a  brownish  color,  when  it  may  be 
examined  (fig.  148).  Instead  of  the  permanganate  one  may  use 
hydrogen  dioxide  (H2O2). 

§415.  Carbon  monoxide  hemoglobin  (CO-hemoglobin). — To 
obtain  this,  kill  an  animal  in  illuminating  gas,  or  one  may  allow 
illuminating  gas  to  bubble  through  some  blood  already  taken  from  the 
body.  The  gas  should  bubble  through  a  minute  or  two.  The  oxy- 
gen will  be  displaced  by  carbon  monoxide.  This  forms  quite  a  stable 
compound  with  hemoglobin,  and  is  of  a  bright  cherry-red  color.  Its 
spectrum  is  nearly  like  that  of  oxy-hemoglobin,  but  the  bands  are 
farther  toward  the  blue.  Add  several  drops  of  ammonium  sulphide 
and  allow  the  blood  to  stand  some  time.  No  reduction  will  take 


262  USING  THE  MICRO-SPECTROSCOPE  [CH.  VIII 

place,  thus  forming  a  marked  contrast  to  solutions  of  oxy-hemoglobin. 
By  the  addition  of  a  few  drops  of  glacial  acetic  acid  a  dark  brownish 
red  color  is  produced. 

§  416.  Carmine  solution.  —  Make  a  solution  of  carmine  by  put- 
ting o.i  gram  of  carmine  in  100  cc.  of  water  and  adding  10  drops  of 
strong  ammonia.  Put  some  of  this  in  a  watch  glass  or  in  a  small 
vial  and  compare  the  spectrum  with  that  of  oxyhemoglobin  or  carbon- 
monoxide  hemoglobin.  It  has  two  bands  in  nearly  the  same  position, 
thus  giving  the  spectrum  a  striking  similarity  to  blood.  If  now  sev- 
eral drops,  15  or  20,  of  glacial  acetic  acid  are  added  to  the  carmine, 
the  bands  remain  and  the  color  is  not  markedly  changed,  while  with 
either  oxy-hemoglobin  or  CO-hemoglobin  the  color  is  decidedly 
changed  from  the  bright  red  to  a  dull  reddish  brown,  and  the  spectrum, 
if  any  can  be  seen,  is  markedly  different.  Carmine  and  O-hemoglobin 
can  be  distinguished  by  the  use  of  ammonium  sulphide,  the  carmine 
remaining  practically  unchanged  while  the  blood  shows  the  single 
band  of  hemoglobin  (§  413).  The  acetic  acid  serves  to  differentiate 
the  CO-tiemoglobin  as  well  as  the  O-hemoglobin. 

§  417.  Colored  bodies  not  giving  banded  spectra.  —  Some  quite 
brilliantly  colored  objects,  like  the  skin  of  a  red  apple,  do  not  give  a 
banded  spectrum.  Take  the  skin  of  a  red  apple,  mount  it  on  a  slide, 
put  on  a  cover-glass,  and  add  a  drop  of  water  at  the  edge  of  the  cover. 
Put  the  preparation  under  the  microscope  and  observe  the  spectrum. 
Although  no  bands  will  appear,  in  some  cases  at  least,  yet  the  ends  of 
the  spectrum  will  be  restricted  and  various  regions  of  the  spectrum 
will  not  be  so  bright  as  the  comparison  spectrum.  Here  the  red 
color  arises  from  the  mixture  of  the  unabsorbed  waves,  as  occurs 
with  other  colored  objects.  In  this  case,  however,  not  all  the  light  of 
a  given  wave  length  is  absorbed;  consequently  there  are  no  clearly 
denned  dark  bands,  the  light  is  simply  less  brilliant  in  certain  regions 
and  the  red  rays  so  predominate  that  they  give  the  prevailing  color. 

§  418.  Nearly  colorless  bodies  with  clearly  marked  absorption 
spectra.  —  In  contradistinction  to  the  brightly  colored  objects  with 
no  distinct  absorption  bands  are  those  nearly  colorless  bodies  and 
solutions  which  give  as  sharply  denned  absorption  bands  as  could  be 
desired.  The  best  examples  of  this  are  afforded  by  solutions  of  the 


CH.  VIII]  USING  THE  MICRO-SPECTROSCOPE  263 

rare  earths,  didymium,  etc.  These  in  solutions  that  give  hardly  a 
trace  of  color  to  the  eye  give  absorption  bands  that  almost  rival  the 
Fraunfoher  lines  in  sharpness. 

§  419.  Absorption  spectra  of  minerals.  —  As  example  take  some 
monazite  sand  on  a  slide  and  either  mount  it  in  balsam  (see  Ch.  X), 
or  cover  and  add  a  drop  of  water.  The  examination  may  be  made 
also  with  the  dry  sand,  but  it  is  less  satisfactory.  Light  well  with 
transmitted  light  and  move  the  preparation  slowly  around.  Absorp- 
tion bands  will  appear  occasionally.  Swing  the  prism  tube  off  the 
ocular,  open  the  slit,  and  focus  the  sand.  Get  the  image  of  one  or 
more  grains  directly  in  the  slit,  then  narrow  and  shorten  the  slit  so 
that  no  light  can  reach  the  spectroscope  that  has  not  traversed  the 
grain  of  sand.  The  spectrum  will  be  satisfactory  under  such  condi- 
tions. It  is  frequently  of  great  service  in  determining  the  char- 
acter of  unknown  mineral  sands  to  compare  the  spectra  with  known 
minerals.  If  the  absorption  bands  are  identical,  it  is  strong  evi- 
dence in  favor  of  the  identity  of  the  minerals.  For  proper  lighting 
see  §  407. 

§  420.  While  the  study  of  absorption  spectra  gives  one  a  great 
deal  of  accurate  information,  great  caution  must  be  exercised  in  draw- 
ing conclusions  as  to  the  identity  or  even  the  close  relationship  of 
bodies  giving  approximately  the  same  absorption  spectra.  The  rule 
followed  by  the  best  workers  is  to  have  a  known  body  as  control  and 
to  treat  the  unknown  body  and  known  body  with  the  same  reagents, 
and  to  dissolve  them  in  the  same  medium.  If  all  the  reactions  are 
identical,  then  the  presumption  is  strong  that  the  bodies  are  identical 
or  very  closely  related.  For  example,  while  one  might  be  in  doubt  be- 
tween a  solution  of  oxy-  or  CO-  hemoglobin  and  carmine,  the  addition 
of  ammonium  sulphide  serves  to  change  the  double  to  a  single  band 
in  the  O-hemoglobin,  and  glacial  acetic  acid  enables  one  to  distinguish 
between  the  CO-blood  and  the  carmine,  although  the  ammonium 
sulphide  would  not  enable  one  to  make  the  distinction.  Further- 
more, it  is  unsafe  to  compare  objects  dissolved  in  different  media. 
Different  objects  as  "cyanine  and  aniline  blue  dissolved  in  alcohol 
give  a  very  similar  spectrum,  but  in  water  a  totally  different  one." 
"Totally  different  bodies  show  absorption  bands  in  exactly  the  same 


264  THE  POLARISCOPE  IN  MICROSCOPY  [Cn.  VIII 

position  (solid  nitrate  of  uranium  and  permanganate  of  potash  in  the 
blue)"  (MacMunn).  The  rule  given  by  MacMunn  is  a  good  one: 
"The  recognition  of  a  body  becomes  more  certain  if  its  spectrum 
consists  of  several  absorption  bands,  but  even  the  coincidence  of  these 
bands  with  those  of  another  body  is  not  sufficient  to  enable  us  to 
infer  chemical  identity,  what  enables  us  to  do  so  with  certainty  is 
the  fact,  that  the  two  solutions  give  bands  of  equal  intensities  in  the 
same  parts  of  the  spectrum  which  undergo  analogous  changes  on  the 
addition  of  the  same  reagent.  It  should  be  borne  in  mind  that  the 
position  of  a  band  may  be  changed  greatly  through  increased  or 
diminished  dissociation,  and  that  the  absorption  bands  given  by  a 
crystal  may  be  quite  different  from  those  given  by  the  same  material 
in  solution  and  furthermore  that  the  absorption  spectra  are  usu- 
ally different  in  different  directions  through  the  crystal"  (Chamot, 
p.  112). 

MlCROPOLARISCOPE 

§  421.  The  micro-polariscope,  or  polarizer,  is  a  polariscope  used 
in  connection  with  a  microscope. 

The  most  common  and  typical  form  consists  of  two  Nicol  prisms, 
that  is,  two  somewhat  elongated  rhombs  of  Iceland  spar  cut  diagonally 
and  cemented  together  with  Canada  balsam.  These  Nicol  prisms 
are  then  mounted  in  such  a  way  that  the  light  passes  through  them 
lengthwise,  and  in  passing  is  divided  into  two  rays  of  plane  polarized 
light.  The  one  of  these  rays  obeying  the  ordinary  law  of  refraction 
is  called  the  ordinary  ray,  the  one  departing  from  the  law  is  called 
the  extraordinary  rays.  These  two  rays  are  polarized  in  planes  at 
right  angles  to  each  other.  The  Nicol  prism  totally  reflects  the  ordi- 
nary ray  at  the  cemented  surface  as  it  meets  that  surface  at  an  angle 
greater  than  the  critical  angle,  and  only  the  less  refracted,  extraordi- 
nary ray  is  transmitted. 

§  422.  Polarizer  and  analyzer.  —  The  polarizer  is  the  Nicol 
prism  placed  beneath  the  object  and  by  means  of  it  the  object  is 
illuminated  with  polarized  light.  The  analyzer  is  the  Nicol  placed 
at  some  level  above  the  object,  very  conveniently  above  the  ocular. 

When  the  corresponding  faces  of  the  polarizer  and  analyzer  are 


CH.  VIII] 


THE   POLARISCOPE   IN   MICROSCOPY 


265 


parallel,  i.e.  when  the  faces  through  which  the  oblique  section  passes 
are  parallel,  light  passes  freely  through  the  analyzer  to  the  eye.     If 


FlG.    150.       MlCRO-POLARISCOPE   IN    POSITION    ON    THE    MICROSCOPE. 

Polarizer     The  Nicol  prism  under  the  stage  of  the  microscope. 

Analyzer     The  Nicol  prism  over  the  ocular. 

Stage     The  stage  of  the  microscope. 

Object     The  object  on  a  slide. 

Objective     The  microscopic  objective. 

S     Set  screw  for  clamping  the  analyzer  to  the  tube  of  the  microscope. 

Ocular     The  microscopic  ocular  in  position. 

Pointer  and  Scale  The  graduated  ring  and  pointer  to  show  the  amount 
of  rotation. 

A  Handle  for  raising  and  lowering  the  analyzer  to  arrange  it  properly 
with  reference  to  the  eye-point. 

these  corresponding  faces  are  at  right  angles,  that  is,  if  the  Nicols 
are  crossed,  then  the  light  is  entirely  cut  off  and  the  two  transparent 
prisms  become  opaque  to  ordinary  light.  There  are  then,  in  the  com- 


266  THE  POLARISCOPE  IN  MICROSCOPY  [Cn.  VIII 

plete  revolution  of  the  analyzer,  two  points  180°  apart  where  the 
corresponding  faces  are  parallel  and  where  light  freely  traverses  the 
analyzer.  There  are  also  two  crossing  points  of  the  Nicols,  midway 
between  the  parallel  positions,  where  the  light  is  extinguished.  In 
the  intermediate  positions  there  is  a  sort  of  twilight. 

§  423.  Putting  the  polarizer  and  analyzer  in  position.  —  Swing 
the  diaphragm  carrier  of  the  condenser  out  from  under  the  condenser, 
open  widely  the  iris  diaphragm,  and  place  the  polarizer  in  the  dia- 
phragm carrier;  then  swing  it  back  under  the  condenser.  Remove 
the  ocular,  put  the  graduated  ring  on  the  top  of  the  tube,  and  then 
replace  the  ocular  and  put  the  analyzer  over  the  ocular  and  ring. 
Arrange  the  graduated  ring  so  that  the  indicator  shall  stand  at  o° 
when  the  field  is  lightest,  or  darkest.  This  may  be  done  by  turning 
the  tube  down  so  that  the  objective  is  near  the  condenser,  then 
shading  the  stage  so  that  none  but  polarized  light  shall  enter  the  micro- 
scope. Rotate  the  analyzer  until  the  lightest  possible  point  is  found, 
then  rotate  the  graduated  ring  until  the  index  stands  at  o°.  The 
ring  may  then  be  clamped  to  the  tube  by  the  side  screw  for  the  pur- 
pose. Or,  more  easily,  one  may  set  the  index  at  o°,  clamp  the  ring 
to  the  microscope,  then  rotate  the  draw-tube  of  the  microscope  till 
the  field  is  lightest,  or  if  the  darkest  point  is  made  zero,  rotate  the 
draw-tube  until  the  field  is  darkest. 

§  424.  Adjustment  of  the  analyzer.  —  The  analyzer  should  be 
capable  of  moving  up  and  down  on  its  mounting,  so  that  it  can  be 
adjusted  to  the  eye-point  of  the  ocular  with  which  it  is  used.  If 
on  looking  into  the  analyzer  with  parallel  Nicols  the  edge  of  the  field 
is  not  sharp,  or  if  it  is  colored,  the  analyzer  is  not  in  the  proper  posi- 
tion with  reference  to  the  eye-point,  and  should  be  raised  or  lowered 
till  the  edge  of  the  field  is  perfectly  sharp  and  as  free  from  color  as 
the  ocular  itself  is  when  the  analyzer  is  removed. 

§  425.  Objectives  to  use  with  the  polariscope.  —  Objectives  of 
all  powers  may  be  used,  including  the  homogeneous  immersion.  In 
general,  however,  the  lower  powers  are  somewhat  more  satisfactory. 
A  good  rule  to  follow  in  this  case  is  the  general  rule  in  all  microscopic 
work,  —  use  the  power  that  most  clearly  and  satisfactorily  shows  the 
object  under  investigation. 


CH.  VIII]  THE  POLARISCOPE  IN  MICROSCOPY  267 

§  426.  Lighting  for  micro-polariscope  work.  —  Follow  the  general 
directions  given  in  Chapter  II.  It  is  especially  necessary  to  shade  the 
object  so  that  no  unpolarized  light  can  enter  the  objective,  other- 
wise the  field  cannot  be  sufficiently  darkened.  No  diaphragm  is 
used  over  the  polarizer  for  most  examinations.  Direct  sunlight  may 
be  used  to  advantage  with  some  objects,  and  the  object  should  be 
as  transparent  as  possible. 

§  427.  Mounting  objects  for  the  polariscope.  —  So  far  as  possible 
objects  should  be  mounted  in  balsam  to  render  them  transparent. 
In  many  cases  objects  mounted  in  water  do  not  give  satisfactory 
appearances  with  the  polariscope.  For  example,  if  starch  is  mounted 
dry  or  in  water,  the  appearances  are  not  so  striking  as  if  mounted 
in  balsam  (Davis,  p.  337). 

§  428.  Purpose  of  a  micro-polariscope.  —  (i)  To  determine 
whether  a  microscopic  object  is  singly  or  doubly  refractive,  i.e., 
isotropic  or  aniso tropic.  (2)  To  determine  whether  or  not  a  body 
shows  pleochroism.  (3)  To  show  whether  an  object  rotates  the 
plane  of  polarization,  as  with  sugar.  (4)  To  give  beautiful  colors. 

For  petrological  and  mineralogical  investigations  the  microscope 
should  possess  a  graduated,  rotating  stage  so  that  the  object  can 
be  rotated  and  the  exact  angle  of  rotation  determined.  It  is  also 
found  of  advantage  in  investigating  objects  with  polarized  light  where 
colors  appear,  to  combine  a  polariscope  and  spectroscope  (spectro- 
polariscope) . 

MICRO-POLARISCOPE  EXPERIMENTS 

§  429.  Arrange  the  polarizer  and  analyzer  as  directed  above 
(§  423)  and  use  a  16  mm.  objective  except  when  otherwise  directed. 

(i)  Isotropic  or  singly  refracting  objects. — Light  the  microscope 
well  and  cross  the  Nicols,  shade  the  stage,  and  make  the  field  as  dark 
as  possible.  For  an  isotropic  substance,  put  an  ordinary  glass 
slide  under  the  microscope.  The  field  will  remain  dark.  As  an 
example  of  crystals  belonging  to  the  cubical  system  and  hence  iso- 
tropic, make  a  strong  solution  of  common  salt  (sodium  chloride),  put 
a  drop  on  a  slide,  and  allow  it  to  crystallize;  put  it  under  the  micro- 
scope, remove  the  analyzer,  focus  the  crystals,  and  then  replace  the 


268  THE  POLARISCOPE  IN  MICROSCOPY  [Cn.  VIII 

analyzer  and  cross  the  Nicols.     The  field  and  the  crystals  will  remain 
dark. 

(2)  Anisotropic    or    doubly    refracting    objects.  —  Make     a    fresh 
preparation  of  carbonate  of  lime  crystals  like  that  described  for  pedesis 
(§  209),  or  use  a  preparation  in  which  the  crystals  have  dried  to  the 
slide ;  use  a  5  or  3  mm.  objective,  shade  the  object  well,  remove  the 
analyzer,  and  focus  the  crystals;  then  replace  the  analyzer.     Cross 
the  Nicols.     In  the  dark  field  will  be  seen  multitudes  of  shining 
crystals,  and  if  the  preparation  is  a  fresh  one  in  water,  part  of  the 
smaller  crystals  will  alternately  flash  and  disappear.     By  observing 
carefully,  some  of  the  larger  crystals  will  be  found  to  remain  dark 
with  crossed  Nicols,  others  will  shine  continuously.     If  the  crystals 
are  in  such  a  position  that  the  light  passes  through  parallel  with  the 
optic  axis  (§  42ga),  the  crystals  are  isotropic  like  salt  crystals  and 
remain  dark.     If,  however,  the  light  traverses  them  in  any  other 
direction,  the  ray  from  the  ^polarizer  is  divided  into  two  constituents 
vibrating  in  planes  at  right  angles  to  each  other,  and  one  of  these 
will  traverse  the  analyzer;  hence  such  crystals  will  appear  as  if  self- 
luminous  in  a  dark  field.     The  experiment  with  these  crystals  from 
the  frog  succeeds  well  with  a  2  mm.  homogeneous  immersion. 

As  a  further  illustration  of  anisotropic  objects,  mount  some  cotton 
fibers  in  balsam  (Ch.  X),  also  some  of  the  lens-paper  (§  158).  These 
furnish  excellent  examples  of  vegetable  fibers;  striated  muscle  fibers 
are  also  very  well  adapted  for  polarizing  objects. 

(3)  Pleochroism.  —  This  is  the  exhibition  of  different  tints  as  the 
analyzer  is  rotated.     An  excellent  subject  for  this  will  be  found  in 
blood   crystals. 

§  429a.  The  optic  axis  of  doubly  refracting  crystals  is  the  axis  along 
which  the  crystal  is  not  doubly  refracting,  but  isotropic  like  glass.  When 
there  is  but  one  such  axis,  the  crystal  is  said  to  be  uniaxial;  if  there  are  two 
such  axes,  the  crystal  is  said  to  be  bi-a?rial. 

The  crystals  of  carbonate  of  lime  from  the  frog  (see  §  209)  are  uniaxiai 
crystals.  Borax  crystals  are  bi-axial. 

§  430.  Starch.  —  One  of  the  most  important  uses  of  a  polariscope 
is  for  the  study  of  starch.  Starch  gives  a  characteristic  black  cross 
which  rotates  as  the  analyzer  is  rotated.  Make  a  thin  slice  of  fresh 
raw  potato  with  a  razor  or  other  sharp  knife  and  mount  it  in  water. 


CH.  VIII]  THE   POLARISCOPE   IN   MICROSCOPY  269 

Use  first  a  16  mm.  and  then  a  higher  power.  The  starch  grains,  many 
of  them,  will  be  found  in  the  potato  cells.  They  have  the  general 
appearance  of  a  clam  or  oyster  shell.  The  black  cross  is  strikingly 
exhibited  by  the  polariscope.  Starch  grains  of  other  plants  show  the 
same,  but  the  grains  are  generally  smaller  and  therefore  do  not  bring 
out  the  structural  features  so  clearly. 

§  431.  Production  of  colors.  —  For  the  production  of  gorgeous 
colors,  a  selenite  plate  is  placed  anywhere  between  the  polarizer  and 
the  analyzer.  If  properly  mounted  the  selenite  is  very  conveniently 
placed  on  the  diaphragm  carrier  of  the  condenser,  just  above  the 
polarizer;  an  unmounted  selenite  may  be  placed  over  the  ocular. 
A  thin  plate  or  film  of  mica  also  answers  well. 

It  is  not  necessary  to  use  selenite  or  mica  for  the  production  of 
vivid  colors  in  many  objects.  One  of  the  most  beautiful  prepara- 
tions and  one  of  the  most  instructive  also,  may  be  prepared  as  follows: 
Heat  some  xylene  balsam  on  a  slide  until  the  xylene  is  nearly  evapo- 
rated. Add  some  crystals  of  the  '  medicine '  sulphonal  and  warm  till 
the  sulphonal  is  melted  and  mixes  with  the  balsam.  While  the 
balsam  is  still  melted  put  on  a  cover-glass.  If  one  gets  perfect  crystals 
there  will  be  shown  beautiful  colors  and  the  black  cross  (Clark). 

It  is  very  instructive  and  interesting  to  examine  many  organic 
and  inorganic  substances  with  a  micro-polarizer. 

COLLATERAL  READING 

Chamot,  Chemical  Microscopy;  Daniell,  Principles  of  Physics;  McMunn, 
The  Spectroscope  in  Medicine. 


CHAPTER   DC 
OPTICS  OF  THE  MICROSCOPE 

§  440.  Apparatus  and  material  for  Chapter  IX. 

1.  Microscope  with  oculars  and  ob-  4.   Homogeneous  immersion  tester, 
jectives.  5.  Ocular    micrometer;     stage    mi- 

2.  Convex  and  concave  lenses.  crometer. 

3.  Apertometer.  6.   Homogeneous      immersion     con- 

denser. 

§  441.    Optical  facts  of  prime  importance  for  the  microscope.  — 
In  considering  the  optics  of  the  microscope  six  fundamental  facts 
concerning    light    must    be   kept   constantly  in    mind,  for   all   of 
them  are  involved  to  a  greater  or  less  degree  in  every  microscopic 
observation: 

(1)  Light  is  composed  of  radiation  which  for  visual  purposes  con- 
sists of  waves  from  Ao.4ju  to  Xo.y/x  in  length. 

(2)  Light  in  a  uniform  medium  extends  in  straight  lines. 

(3)  Light  may  be  reflected. 

(4)  Light  is  refracted  in  passing  from  one  medium  to  another  of 
different  density. 

(5)  Light  may  be  dispersed  or  grouped  into  colored  rays  from  the 
fact  that  rays  of  different  wave  length  are  differently  bent  (fig.  145,  2). 

(6)  Light  may  be  diffracted. 

Stated  in  briefest  terms  light  exhibits  the  properties  of: 
(i)   Wave  motion;    (2)    Rectilinear  motion;    (3)    Reflection;    (4) 
Refraction;    (5)   Dispersion;    (6)   Diffraction. 

§  442.  Wave  motion.  —  From  a  body  like  the  sun,  the  electric 
arc  and  other  sources  of  energy,  radiations  are  given  off  in  waves. 
The  radiation  which  is  visible,  forms  but  a  very  small  segment  of  the 
total  radiation.  In  fig.  151  the  visible  radiation  is  shown  between 
wave  lengths  Xo.4/x  and  Ao.y/z,  measured  in  air  or  in  a  vacuum. 
Shorter  waves  are  called  ultra-violet,  and  longer  waves  infra-red. 

270 


CH.  IX]  VISIBLE    AND    INVISIBLE    RADIATION  271 

The  infra-red  waves  are  only  shown  up  to  a  length  of  2^,  although 
many  of  much  greater  length  exist. 

In  the  ether  of  space  the  different  visible  waves  move  with  equal 
velocity,  but  in  the  various  transparent  bodies  on  the  earth,  the  ve- 
locity depends  upon  the  wave  length  —  the  shorter  the  waves  the 
slower  the  motion  (§  451). 

§  443.  Light  moves  in  straight  lines.  —  In  a  uniform  medium 
light  moves  in  straight  lines.  Any  body  in  which  light  can  move 

A  2, 


Visible 
Radiation 
Ultra-Violet  Infra-Red 

FIG.  151.    VISIBLE  AND  INVISIBLE  RADIATION. 

The  segment  in  white  shows  the  visible  radiation,  which  lies  between 
and  Xo.7/z.  Radiation  shorter  than  Ao.4ju  is  known  as  ultra-violet,  and  longer 
than  Xo.7ju  as  infra-red.  This  is  a  normal  spectrum  magnified  20,000  vertically 
and  50,000  times  horizontally  (sep  also  fig.  144). 

freely  is  said  to  be  transparent.  If  light  meets  a  body  in  which  it 
cannot  move  it  is  either  reflected  (§  444)  or  absorbed;  if  absorbed 
it  is  changed  to  some  other  form  of  energy,  usually  heat. 

§  444.  Reflection. —  If  light  meets  a  surface  which  is  opaque  or 
only  partly  transparent,  it  is  changed  in  its  course  or  reflected;  or 
it  may  be  absorbed. 

If  the  surface  is  smooth  and  the  light  is  reflected,  the  incident  and 
the  reflected  ray  will  be  in  the  same  plane  and  will  make  equal  angles 
on  opposite  sides  of  a  normal  erected  at  the  point  of  reflection  (fig.  152). 
The  eye  can  see  the  light  only  when  in  the  path  of  the  ray,  or  when 
light  is  deflected  from  the  ray  by  dust,  etc.  (§  117). 


272 


REFLECTION    AND    REFRACTION    OF    LIGHT       [CH.  IX 


If  the  surface  is  irregular  the  reflection  will  also  be  irregular  and 
the  light  will  be  reflected  from  the  point  of  incidence  in  the  form  of  a 

hemisphere  (fig.  153),  hence 
light  would  reach  the  eye  from 
any  point  in  the  hemisphere. 
§  446.  Refraction.  —  As  or- 
dinarily considered,  this  is  the 
change  in  direction  which  light 
undergoes  when  passing  ob- 
liquely from  one  transparent 
medium  into  another  (fig.  1 54- 


FIG.  152.    REGULAR  OR  MIRROR 
REFLECTION. 

(From  Optic  Projection). 


The  angle  of  incidence  i,  is  equal  to  the 
angle  of  reflection  r\  and  the  incident  and 
reflected  ray  are  in  a  plane  perpendicular  to 
the  reflecting  surface. 


A  broader  statement  cover- 
ing all  the  phenomena  whether 
the  ray  passes  obliquely  or 
normally  from  one  medium  to 
another  is  this:  Refraction  is  the  change  in  velocity  of  the  waves  of 
light  in  passing  from  one  transparent  medium  into  another. 

§  446.  Law  of  refraction.  —  The  amount  of  bending  depends  upon 
two  factors,  —  the  relative  density  of  the  two  media  and  the  obliquity 
of  the  incident  light.  The  greater  the  ob- 
liquity of  the  incident  ray,  and  the  greater 
the  difference  in  density,  the  greater  will 
be  the  refraction.  The  precise  law  govern- 
ing the  course  and  relation  of  the  ray  in 
the  two  media  is  known  as  the  sine  law  of 
Snell  and  Descartes.  It  is  expressed  thus: 


sin.  i 
sin  r 


=  index  of  refraction.     That  is,  the 


sine  of  the  angle  of  the  incident  ray  with 

the  normal,  divided  by  the  sine  of  the  angle 

of  the  refracted  ray  with  its  normal,  gives 

the  relative  direction  of  the  ray  in  the  two 

media,  i.e.,   the  index   of  refraction.     For 

example  in  fig.  154,  showing  the  passage  of  light  to  water,  the  ray 

being  at  60°  with  the  normal  in  air,  and  40°  38'  in  water,  the  real 


FIG.   153.     IRREGULAR  OR 
DIFFUSE  REFLECTION'. 

(From  Optic  Projection). 

A  ray  of  light  meeting  a 
rough  surface,  like  a  piece  of 
white  paper,  is  scattered  al- 
most equally  in  all  direc- 
tions, making  a  hemisphere 
of  light. 


CH.  IX] 


REFRACTION    OF    LIGHT 


273 


relationship  in  this  and  in  all  other  cases  is  not  the  relative  size  of  the 

sin  i  or  0.86603 

two  angles,  but  the  sines  of  the  angles,  thus:   -  -  =  1.33. 

smr  or  0.65115 

That  is,  the  sine  of  the  angle  in  air  is  1.33  times  the  sine  of  the  angle 

in  water;  and  this  would  hold 

true  for  any  other  pair  of  sines, 

so  that  the  law  is  universal  for 

the  wave  length  of  light  giving 

this  index  of  refraction. 
The  sine  and  corresponding 

angle  are  always  greater  in  the 

rarer  medium  and  consequently 

less  in  the  denser  medium.     It 

follows  from  this  that  when  the 

ray  passes  from  a  rarer  to   a 

denser  medium  and   the  angle 

is  made  less,  the  ray  must  bend 

toward  the  normal.     Conversely 

in  passing  from  a  denser  to  a 

rarer  medium  where  the  angle 

is  greater,  the  ray  must  bend  from  the  normal.     This  is  a  general 

law  (see  fig.  155,  157). 

§  447.  Absolute  index  of  re- 
fraction. —  This  is  the  index  of 
refraction  obtained  when  the  in- 
cident ray  passes  from  a  vacuum 
into  a  given  medium.  As  the 
index  of  the  vacuum  is  taken  as 
unity,  the  absolute  index  of  any 
substance  is  always  greater  than 
unity.  For  many  purposes,  as 
for  the  object  of  this  book,  air 
is  treated  as  if  it  were  a  vacuum, 
and  its  index  is  called  unity,  but 
in  reality  the  index  of  refraction 
of  air  is  about  3  ten-thousandths 


FIG.  154.     REFRACTION  OF  LIGHT  IN 
PASSING  FROM  AIR  TO  WATER. 

N  Normal  at  the  point  of  refraction. 
sin  i    In  this  example  sin  60°  or  0.86603 
sin  r   In  this  case  sin  40°  38'  or    0.65115 
=  1.33,  average  index  of  refraction  for  air 
and  water. 


FIG.  155.    REFRACTION  OF  LIGHT  IN 


PASSING  FROM  AIR  TO  GLASS. 

N  Normal  at  the  point  of  refraction. 
sin  i  In  this  example  sin  60°  or  0.86603 
sin  r  In  this  example  sin  34°  45'  or  0.56975 
=  1.52,  average  index  of  refraction  for  air 
and  glass. 


274 


INDEX    OF    REFRACTION 


[Cn.  IX 


greater  than  unity.  Whenever  the  refractive  index  of  a  substance  is 
given,  the  absolute  index  is  meant  unless  otherwise  stated.  For  ex- 
ample, when  the  index  of  refraction  of  water  is  said  to  be  1.33,  and 
of  crown  glass  1.52,  etc.,  these  figures  represent  the  absolute  index, 
and  the  incident  ray  is  supposed  to  be  in  a  vacuum. 

§  448.  Relative  index  of  re- 
fraction. —  This  is  the  index  of 
refraction  between  two  contigu- 
ous media,  as  for  example  be- 
tween glass  and  diamond,  water 
and  glass,  etc.  It  is  obtained 
by  dividing  the  absolute  index 
of  refraction  of  the  substance 
containing  the  refracted  ray,  by 
the  absolute  index  of  the  sub- 
stance transmitting  the  incident 
ray.  For  example,  the  relative 
index  from  water  to  glass  is  1.52 
divided  by  1.33.  If  the  light 
passed  from  glass  to  water  it 
would  be,  1.33  divided  by  1.52. 
By  a  study  of  the  figures 


FIG.  156.    REFRACTION  OF  LIGHT  IN 
PASSING  FROM  GLASS  TO  AIR. 

N    Normal  to  the  refracting  surface. 
sin  i    In  this  case  sin  34°  45'  or  0.56975 
sin  r    In  this  case  sin  60°       or  0.86603 

i_ 

1.52 


If  fig.  155  and  156  are  compared  it  will 
be  seen  that  the  ray  of  light  follows  exactly 
the  same  path  in  leaving  the  denser  me- 
dium that  it  took  on  entering  it. 


showing  refraction,  it  will  be 
seen  that  the  greater  the  re- 
fraction the  less  the  angle  and  consequently  the  less  the  sine  of 
the  angle,  and  as  the  refraction  between  two  media  is  the  ratio 

of  the  sines  of  the  angles  of  incidence  and  refraction  ( -   - ),  it  will 

\sin  r' 

be  seen  that  whenever  the  sine  of  the  angle  of  refraction  is  increased 
by  being  in  a  less  refractive  medium,  the  index  of  refraction  will  show 
a  corresponding  decrease  and  vice  versa.  That  is,  the  ratio  of  the  sines 
of  the  angles  of  incidence  and  refraction  of  any  two  contiguous  substances 
is  inversely  as  the  refractive  indices  of  those  substances.  The  formula  is: 


(Sine  oi 
Sine  of 


of  angle  of  incident  ray\      /Index  of  refraction  of  refracting  medium 


angle  of  refracted  ray/      \Index  of    refraction  of    incident  medium 


CH.  IX]       CRITICAL    ANGLE    AND    TOTAL    REFLECTION  275 

,  /sin  A      /index  r\ 

Abbreviated  (  — I  =  I      :  1.      By   means    of    this   general    formula   one 

\sin  r)      \index  ij 

can  solve  any  problem  in  refraction  whenever  three  factors  of 
the  problem  are  known.  The  universality  of  the  law  may  be  illus- 
trated by  the  following  cases: 

(A)  Light  incident  in  a  vacuum  or  in  air,  and  entering  some  denser 
medium,  as  water,  glass,  diamond,  etc. 

/  Sine  of  angle  made  by  the  ray  in  air  \  _  /Index  of  ref.  of  denser  medA 
\Sine  of  angle  made  by  ray  in  denser  med./  \  Index  of  ref.  of  air  (i)  / 

If  the  dense  substance  were  glass  (  —   —)  =  (-—).     If  the  two  media  were 

\sin  rj      \   i    / 

water  and  glass,   the  incident  light  being  in   water   the   formula  would  be: 

(—  J  =  I  _  —  J.      If  the  incident  ray  were  glass  and  the  refracted  ray  in 
sin  rj      \i.33/ 

water:   (  —   —  J  =  (  -----  ).      And  similarity  for  any  two  media;   and  as  stated 
Vsin  rj      \i.SV 

above  if  any  three  of  the  factors  are  given  the  fourth  may  be  readily  found. 

§  449.  Critical  angle  and  total  reflection.  —  In  order  to  under- 
stand the  Wollaston  camera  lucida  (fig.  99)  and  other  totally  reflect- 
ing apparatus,  it  is  necessary  briefly  to  consider  the  critical  angle. 

The  critical  angle  is  the  greatest  angle  that  a  ray  of  light  in  the 
denser  of  two  contiguous  media  can  make  with  the  normal  and  still 
emerge  into  the  less  refractive  medium.  On  emerging  it  will  form  an 
angle  of  90°  with  the  normal,  and  if  the  substances  are  liquids,  the 
refracted  ray  will  be  parallel  with  the  surface  of  the  denser  medium. 

Total  Reflection.  —  In  case  the  incident  ray  in  the  denser  medium 
is  at  an  angle  with  the  normal  greater  than  the  critical  angle,  it  will 
be  totally  reflected  at  the  surface  of  the  denser  medium,  that  surface 
acting  as  a  perfect  mirror.  By  consulting  the  figures  it  will  be  seen 
that  there  is  no  such  thing  as  a  critical  angle  and  total  reflection  in 
the  rarer  of  two  contiguous  media. 

To  find  the  critical  angle  in  the  denser  of  two  contiguous  media:  — 

Make  the  angle  of  refraction  (i.  e.,  the  angle  in  the  rarer  of  the  two 

j.  N      0  /sin  i\      /index  r\ 

edia)  90   and  solve  the  general  equation:  ( )  =  I ). 

\sin  rj      Vindex  iJ 


m 


276 


CRITICAL    ANGLE    AND    TOTAL    REFLECTION       [Cn.  IX 


(i)    Critical    angle    of  water  and  air:   sin  r  (90°)  is   i,  index   of 

water  1.33  whence  (  -  -)  =  ( )  or  sin  i  =  751+.  This  is  the  sine 

\  i  '  M-33' 

of  48°  45',  and  whenever  the  ray  in  the  water  is  at  an  angle  of  more 

than  48°  45'  it  will  not  emerge 
into  the  air,  but  be  totally  re- 
flected back  into  the  water. 

(2)  Critical  angle  of  glass  and 
air:   sin  r  (90°)   is   i.     index  for 

,       .                         sin  i        i 
glass  is  i. 5 2,  whence =  - 

i        1.52 

sin  0.65789,  which  is  the  sine  of 
41°*.  Light  having  a  greater 
angle  in  glass  than  41°  is  intern- 
ally reflected  as  from  a  mirror 
(fig.  152),  and  reflected  back  into 
the  glass. 

(3)  Critical     angle     of     glass 
covered  with  water. 


Air 


\ 

=  i' 


sin  r  (sin  90°  =  i) 
Andex  water  (1.33)^        /'sin  i 
>>  index  glass  (1.52)  ' 


rv- 


FIG.  157.  DISPLACEMENT  OF  A  RAY 
OF  LIGHT  IN  TRAVERSING  AN  OBJECT 
WITH  PLANE  FACES. 

This  figure  is  to  show  that  while  there 
is  no  angular  deviation  of  a  ray  of  light 

in  traversing  a  dense  medium  with  plane  f  •  Sin  i 

faces,  there  is  displacement;  but  the  em- 
erging ray  (r)  is  parallel  with  the  entering 
ray  (i). 

Air  Glass  The  two  media  through 
which  the  ray  is  traveling. 

/  n  Incident  ray  and  normal  at  the 
point  of  entrance  into  the  glass. 

i'  Incident  ray  continued  by  dotted 
lines  to  show  the  path  which  would  have 
been  followed  if  no  glass  had  intervened.  wnence  sin  /  =  .875  sine  of  critical 

n  r  Normal  and  refracted  ray  on  em- 
ergence from  the  glass  to  the  air  again,  angle  in  glass  covered  with  water. 

r'  Path  of  the  refracted  ray  traced  The  corresponding  angle  is  ap- 
backward. 

proximately  61  . 

The  last  shows  the  advantage  of  water  immersion  when  a  large 
angle  of  light  is  desired.  With  homogeneous  immersion  there  would 
be  no  critical  angle  for  the  glass. 

§  44pa.  Critical  angle.  —  As  defined  by  some  physicists  the  critical  angle  is  the 
least  angle  at  which  light  undergoes  total  internal  reflection  at  the  surface  of  the 
denser  medium. 


CH.  IX]          INDEX    OF    REFRACTION    AND    VELOCITY 


277 


I  have  followed  the  more  common  definition  which  makes  it  the  greatest  angle 
?t  which  a  ray  can  emerge  into  the  rarer  medium;  the  emerging  angle  will  then  be 
90°  and  its  sine  i.ooo. 

§  450.   Table  of  refractive  indices.    (From  Chamot). 
(Temperature  20  to  22  C.) 


Index  of 
Refraction 

Xame  of 
Substance 

Approximate 
Boiling 
Point  °C 

Approximate 
Density 

1.32 

Methyl  alcohol 

66 

0.79 

1.30 

Ethyl  ether 

35 

0.71 

i-37 

Ethyl  alcohol 

78 

0.79 

1.46 

Cajeput  oil 

i?4 

0.92 

1.44 

Chloroform 

61 

1.48 

i-47 

Glycerine 

290 

1.61 

1.47 

Turpentine 

155 

0.86 

.48 

Castor  oil 

0.96 

49 

Xylene 

136' 

0.86 

•49 

Benzene 

80 

0.88 

•5° 

Clove  oil 

1.05 

•5r 

Cedar  Wood  oil 

0.98 

•57 

Orthotoluidine 

197 

I.OO 

.625 

Carbon  bisulphide 

46 

1.29 

-52± 

Canada  balsam 

.52-1-59 

Glass 

.... 

•544-1-553 

Quartz 

§  451.  The  sine  law  and  the  velocity  of  light  in  different  media.— 
In  the  ether  of  space  all  wave  lengths  of  light  move  with  equal  velocity, 
but  on  the  earth  the  velocity  depends  on  the  wave  length.  While  all 
wave  lengths  are  retarded  by  shortening  the  waves,  the  shorter  the 
original  wave  the  greater  the  retardation.  As  the  refraction  of  the 
light  is  one  of  the  phenomena  of  this  retardation  it  follows  that 
the  shorter  the  wave  the  greater  the  bending.  This  is  shown  by  the 
action  of  the  prism  (fig.  145,  2),  in  which  the  blue  is  more  deviated 
than  the  red. 

The  retardation  of  any  given  wave  length  (i.e.  the  relative  shorten- 
ing of  the  waves)  follows  the  sine  law  in  passing  from  one  transparent 
substance  to  another.  For  example,  in  passing  from  the  ether  to 

water  the  speed  in  water  would  be  represented  by:  -    -  or  1.334  for 

sin  r 


278 


INDEX  OF  REFRACTION  AND  VELOCITY    [Cn.  IX 


waves  at   the  D   Fraunhofer   line   (Watson).     This  means  that  if 
the  speed  in  the  ether  were  i,  that  in  water  for  this  wave  length  the 


velocity  would  be 


In  terms  of  the  angle  of  the  light,  if  the 


1-334 
sine  of  the  angle  in  the  ether  is  i,  the  sine  of  the  angle  of  this  wave 

i 


length  in  water  would  be 


1-334 


FIG.  159. 


FIG.  158. 

FIG.  158.     CRITICAL  ANGLE  FOR  LIGHT  PASSING  FROM  WATER  TO 
AIR,  THE  ANGLE  IN  AIR  BEING  90°. 

N    Normal  to  the  refracting  surface. 

sini  In  this  case  sin       4f  45'  or  0.7510  =  _i_    in  accordance  with  the  general 
Sin  r  In  this  case  sin      90  or  I>0000      I>33' 

formula:  ™1  =  i-^-r 
sin  r     index  i 

b  Light  ray  at  the  critical  angle  and  emerging  into  the  air  parallel  with  the 
surface  of  the  water. 

d  dr  Ray  of  light  at  an  angle  greater  than  the  critical  one  and  being  internally 
reflected  back  into  the  water;  the  angle  of  incidence  and  reflection  being  equal 
(fig.  152). 

FIG.  159.    CRITICAL  ANGLE  FOR  LIGHT  PASSING  FROM  GLASS  TO 
AIR,  THE  ANGLE  IN  AIR  BEING  90°. 

N    Normal  to  the  refracting  surface. 

sinj.    In  this  case  sin  41°  +  or  0.65789  =_^_      in    accordance    with    the   general 
sin  r  In  this  case  sin  90       or  1>oooo        I<52' 


formula: 

sin  r      index  i 

b    Light  ray  at  the  critical  angle  and  emerging  into  the  a'ir  parallel  with  the 
surface  of  the  glass. 

d  df     Ray  of  light  at  an  angle  greater  than  the  critical  angle  and  being  reflected 
back  into  the  glass,  the  angle  of  incidence  and  reflection  being  equal  (Fig.  152). 


CH.  IX]          INDEX    OF    REFRACTION    AND    VELOCITY  279 

For  crown  glass  the  waves  opposite  the  fixed  line  J5,  if  possessed  of 
a  speed  of  i  in  the  ether,  would  have  a  speed  in  the  glass  of  - 

Opposite  the  H  line,  with  the  shorter  waves,  the  speed  would  be  - 


in  crown  glass. 

That  is,  then,  just  as  in  refraction  (§  445-446),  if  the  velocity  in 
one  medium  and  the  index  of  refraction  of  the  two  media  are  known 
the  velocity  in  the  second  me- 
dium can  be  determined;  and 
in  general  knowing  any  three 
factors  the  fourth  can  be  de- 
termined. 

While  for  the  discussion  of 
lenses  the  narrower  view  of  re- 
fraction may  suffice,  for  optical 
instruments  generally  it  is  of 
fundamental  importance  to  real- 
ize that  there  is  just  as  much 
effect  on  light  waves  striking 
the  surface  of  the  refracting 
body  perpendicularly  as  ob- 
liquely. In  one  case,  that  of  the 
oblique  meeting,  the  ray  is  bent 
due  to  the  shortening  of  the 
waves  in  passing  from  a  rarer 
to  a  denser  medium.  If  the 
waves  meet  the  denser  sub- 
stance normally  to  its  surface 
the  ray  will  not  be  bent,  but  the 
shortening  of  the  waves  will  be 
the  same  leading  to  an  optical 
shortening  of  the  path  of  the  ray.  This  is  of  prime  value  when  de- 
signing optical  apparatus  where  two  optical  paths  must  be  made 
equal,  although  the  actual  distance  in  millimeters  may  be  unequal. 
The  binocular  microscope  is  a  striking  example  (fig.  53-54).  The 


FIG.  1 60.  CRITICAL  ANGLE  FOR  LIGHT 
PASSING  FROM  GLASS  TO  WATER,  THE 
ANGLE  IN  THE  WATER  BEING  90°. 


N 
sin  i 


Normal  to  the  refracting  surface. 
In  this  case  sin  61°  +  or  0.8750  _ 
sin  r  In  this  case  sin  90° 


or  i.oooo 

— ^  in  accordance  with  the  general  form- 
index  r 


1.52 


ula 


sin  ^ 


sin  r     index  i 

b  Light  ray  at  the  critical  angle  and 
emerging  into  the  water  at  an  angle  of 
90°  from  the  normal. 

d  d'  Ray  of  light  at  an  angle  greater 
than  the  critical  angle  and  hence  reflected 
back  into  the  glass,  the  angle  of  incidence 
and  reflection  being  equal. 


280  DISPERSION    AND    DIFFRACTION  [Cn.  IX 

shortening  of  the  path  is  also  very  strikingly  illustrated  by  the  cover- 
glass  (fig.  31-32,  §  80-81). 

§  4&2.  Dispersion.  —  This  is  the  separation  of  waves  of  composite 
light  into  groups  which  appear  of  different  colors  to  the  normal  eye. 
If  white  light  is  dispersed  there  results  the  familiar  rainbow,  or 
spectrum. 

As  this  dispersion  is  due  to  the  different  refrangibility  of  the  different 
wave  lengths,  the  shortest  waves  being  most  bent,  one  would  expect 
that  the  amount  of  bending  would  be  in  exact  proportion  to  the  wave 
lengths.  This  is  true  if  one  uses  a  diffraction  grating  and  forms  a 
normal  spectrum  (fig.  146).  When  a  prism  is  used  to  produce  the 
dispersion  (145,  2),  the  dispersion  is  not  in  exact  relation  to  the  wave 
length.  In  general  the  red  end  of  the  spectrum  is  condensed  and  the 
blue  end  expanded  (fig.  147,  148-149).  Different  kinds  of  glass  dis- 
perse differently  and  the  same  is  true  of  transparent  minerals,  quartz, 
fluorite,  etc.  This  makes  achromatism  possible.  As  pointed  out  by 
Newton,  if  the  dispersion  were  in  exact  proportion  to  the  wave  length 
as  with  gratings,  whenever  dispersion  is  overcome,  refraction  would 
also  be  overcome  and  no  achromatic  combination  of  lenses  would  be 
possible. 

§  453.  Diffiraction.  —  This  is  the  bending  of  light  past  the  edge  of 
objects.  Instead  of  the  light  all  going  in  a  straight  line  beyond  an 
object,  especially  a  narrow  strip,*  some  of  it  extends  as  if  split  off 
from  the  main  beam  at  the  edge  of  the  obstruction.  These  dif- 
fracted beams  may  give  rise  to  independent  or  so-called  spurious 
images.  With  low  powers  the  diffracted  light  does  not  cause  com- 
plications, but  with  high  powers  the  diffraction  fringes  and  diffrac- 
tion disc  may  produce  effects  very  difficult  of  interpretation.  (See 
§  474  where  there  is  a  discussion  of  the  part  played  by  diffracted 
light  in  microscopic  images.) 

LENSES  AND  IMAGES 

§  454.  Lenses.  —  A  lens  is  a  transparent  body  having  one  or  both 
of  its  opposite  sides  curved.  The  curves  are  most  frequently  spherical, 
and  may  be  either  convex  or  concave.  If  both  the  surfaces  are  curved 
the  lens  may  be  considered  as  composed  of  segments  of  two  spheres. 


CH.  IX] 


IMAGES    FORMED    BY    LENSES 


281 


These  spheres  are  of  like  radius 
if  the  surfaces  are  similarly 
curved,  and  of  unlike  radius  if 
the  surfaces  are  unlike.  While 
a  lens  with  one  plane  face  may 
be  considered  a  segment  of  a 
single  sphere,  optically  it  is 
better  to  consider  two  spheres, 
the  curved  surface  from  a 


FIG.  161-162.    A  CONCAVE  LENS  Snow- 


aim 


sphere  of  finite,  and  the  plane   ING  THE  PRINCIPAL,  VIRTUAL  Focus;  AND 
e         e  e  •  ^    •,      CONVEX  LENS  SHOWING  THE  REAL  PRIN- 

face  from  a  sphere  of  infinite  CIPAL  Focus  (F  F) 

radius  (fig.  167,  3,  6). 

§  455.  Images  formed  by  lenses.  —  As  light  entering  a  dense  trans- 
parent body  obliquely  is  bent  toward 
the  normal  at  the  point  of  entrance,  it 
follows  that  if  the  lens  has  convex  faces 
the  light  rays  will  be  made  more  con- 
vergent; if  it  has  concave  faces  the 
light  rays  will  be  rendered  more  diverg- 
ent (fig.  161-162).  From  the  change 
in  the  direction  of  the  rays  on  entering 
and  on  leaving  a  lens  it  is  possible  to 
form  images  of  objects  by  means  of 
lenses  (fig.  163-166). 

§  456.  Forms  and  principal  features 
of  spherical  lenses.  —  As  shown  in  fig. 
167  lenses  may  be  convex  on  both  faces, 
or  convex  on  one  face  and  plane  or  con- 
cave on  the  other.  Lenses  may  also  be 
concave  on  both  faces  or  concave  on 
one  face  and  plane  or  convex  on  the 
other. 

If  lenses  are  thick  in  the  middle  and 
thin  on  the  edge,  they  make  the  rays 
of  light  entering  them  more  converg- 
ent. On  the  other  hand,  if  they  are 


FIG.  163-164.  To  SHOW  THE 
FORMATION  or  A  REAL  AND  or 
A  VIRTUAL  IMAGE  BY  A  CONVEX 
LENS.  (COMPARE  FIG.  11-12). 


The  size  of  the  image  depends 
upon  its  relative  distance  from 
the  center  of  the  lens.  If  it  is 
farther  from  the  center  than  the 
object  it  will  be  larger  than  the 
object,  but  if  nearer  it  will  be 
smaller  (fig.  84). 


282 


IMAGES    FORMED    BY    LENSES 


[CH.  IX 


thin  in  the  middle  and  thick  on  the  edge  they  make  the  light  rays 
entering  them  more  divergent.  In  a  word,  then,  thin  edge  lenses 
are  called  convergent,  and  thick  edge  ones,  divergent  lenses.  This 
follows  inevitably  from  the  rule  that  on  entering  a  denser  medium 


FIG.  165-166.  To  SHOW  THE  FORMATION  OF  A  REDUCED  VIRTUAL  IMAGE  BY 
A  CONCAVE  LENS,  AND  THAT  THE  IMAGE  is  LARGER  THE  NEARER  THE  OBJECT 
Is  TO  THE  PRINCIPAL  (VIRTUAL)  Focus.  (COMPARE  FIG.  86-87). 

any  oblique  ray  of  light  is  bent  toward  the  normal,  and  on  leaving 
it  for  a  rarer  medium,  it  is  bent  from  the  normal  (§  446.) 

§  457.  Principal  features  of  spherical  lenses  —  (i)  Principal 
axis.  This  is  the  straight  line  passing  through  the  lens  and  joining 
the  centers  of  the  two  spheres  contributing  to  the  formation  of  the 
lens  (fig.  167,  40  c'). 

(2)  Optic  center.  The  point  in  a  lens  or  near  it  through  which 
light  rays  pass  without  angular  deviation.  That  is,  the  ray 


CH.  IX]  LENSES    AND    THEIR    PRINCIPAL    FORMS  283 

\  23 


FIG.  167. 


SPHERICAL  LENSES  WITH  THEIR  FORMS  AND  PRINCIPAL 
FEATURES. 


(1)  Double  convex  lens  showing  the  two  spheres  from  which  it  was  derived. 
c-c'  the  centers  of  the  two  spheres  with  the  principal  axis  of  the  lens  on  the  line 
joining  the  centers. 

(2)  Double  concave  lens  and  the  two  spheres  from  which  it  was  derived,     c-c' 
centers  of  the  spheres  and  axis  of  the  lens. 

(3)  Plano-convex  lens  with  the  sphere  from  which  it  was  derived.     In  this  case 
the  axis  is  on  the  radius  dividing  the  lens  into  two  equal  parts. 

(4)  Double  convex  lens  showing  the  two  spheres  from  which  it  was  derived; 
r  r'  parallel  radii;   /  /'  tangents  at  the  ends  of  the  radii;   c  c'  centers  of  the  two 
spheres,  on  the  connecting  line  of  which  is  the  principal  axis  of  the  lens,  and  the 
optic  center  (d). 

(5)  Double  concave  lens  showing  the  same  features  as  in  (4). 

(6)  Plano-convex  lens  showing  the  same  as  in  (5).     In  this  case  the  radius  of 
the  curved  face  is  determined  as  usual,  but  that  of  the  plane  face  may  be  considered 
infinity,  so  that  any  line  perpendicular  to  the  plane  face  is  a  part  of  that  radius. 
As  shown  in  the  figure  the  center  of  the  lens  must  be  then  at  the  convex  surface 
of  the  lens. 

(7)  Plano-concave  lens  the  parts  are  practically  like  (6). 

(8)  Thin  edge  or  converging  meniscus  lens  with  the  two  spheres  from  which 
it  was  derived.     The  inner,  concave  face  is  from  the  greater  sphere,  and  the 
optic  center  (d)  is  wholly  outside  the  lens. 

(9)  Thick  edge  or  diverging  meniscus  lens.     In  this  case  the  concave  face  is 
from  the  smaller  sphere,  and  the  center  of  the  lens  (d)  is  on  the  concave  side. 


284  ABERRATION  OF  LENSES  [Cn.  IX 

passing  through  the  center  of  the  lens  continues  in  a  line  parallel  to 
the  original  direction  as  it  does  in  traversing  a  piece  of  plane  glass 

(%  157). 

As  shown  in  the  diagrams  (fig.  167)  the  optic  center  is  found  by 
drawing  parallel  radii  from  the  two  curved  surfaces,  or  from  the  curved 
and  plane  surface,  and  joining  the  ends  of  the  radii.  The  center  of 
the  lens  is  at  the  point  where  a  line  connecting  the  ends  of  the  radii 
crosses  the  principal  axis  (fig.  167,  cf).  The  reason  why  light  rays 
traversing  the  optic  center  have  no  angular  deviation  is  evident, 
for  the  radii  are  perpendicular  to  the  surface  of  the  lens,  and  the 
tangent  plane  perpendicular  to  the  radius  is  tangent  to  the  sphere 
at  the  end  of  the  radius.  As  the  tangents  of  two  parallel  radii  must 
themselves  be  parallel,  it  follows  that  a  ray  of  light  passing  from  one 
tangential  point  to  the  other  is  traversing  a  body  with  parallel 
sides  at  the  point  of  entrance  and  exit,  and  hence  it  will  suffer  no- 
angular  deviation.  The  ray  may  be  displaced  as  in  traversing  any 
thick  transparent  body  (fig.  157).  With  meniscus  lenses  the  optic 
center  (fig.  167,  8,  9)  is  on  an  extension  of  the  line  joining  the  centers 
of  curvature,  and  wholly  outside  the  lens. 

(3)  Secondary  axis.   This  is  any  line  which  passes  through  the 
optic  center  of  the  lens  and  is  oblique  to  the  principal  axis. 

(4)  Principal  focal  point.   The  principal  focal  point  or  focus  of 
a  lens  or  of  a  lens  system  like  an  objective,  a  simple  microscope,  etc., 
is  the  point  on  the  principal  axis  where  rays  of  light  parallel  to  the 
principal  axis  before  entering  the  lens  or  lens  system,  cross  the  prin- 
cipal ^axis  after  leaving  the  lens  or  objective  (fig.   161-162).     The 
focus  is  also  called  the  burning  point.     With  a  concave  mirror  it  is 
the  point  on  the  principal  axis  where  rays  parallel  with  the  principal 
axis  before  meeting  the  mirror,  cross  the  principal  axis  after  reflec- 
tion from  the  concave  surface.     This  point  is  situated  half-way  be- 
tween the  face  of  the  mirror  and  the  center  of  curvature. 

ABERRATION  OF  LENSES 

§  458..  Spherical  aberration.  —This  is  a  defect  of  spherical  lenses 
shown  in  fig.  168.  That  is,  the  parallel  ray  at  the  edge  crosses  the 
principal  axis  or  comes  to  a  focus  nearer  the  center  of  the  lens  than  a 


CH.  IX]      CORRECTION  OF  THE  ABERRATIONS         285 

ray  near  the  axis.  If  then  the  full  aperture  is  filled,  as  shown  in  the 
figure,  with  rays  parallel  with  the  axis,  there  will  be  a  series  of  foci, 
those  of  the  border  rays  being  nearer  the  lens  than  those  near  the 
middle  of  the  lens  (fig.  168,  fi,  £2,  13). 

§  459.  Correction  of  spherical  aberration.  —  It  is  possible  by 
selecting  convex  and  concave  lenses  of  different  material  and  hence 
of  different  refractive  power,  to  overcome  the  spherical  aberration 
of  the  convex  lens  with  an  equal  and  opposite  aberration  in  a  concave 
lens  without  overcoming  the  converging 
action  of  the  convex  lens.  Consequently 
rays  will  all  come  to  one  focus.  Such  a 
lens  combination  is  said  to  be  aplanatic 
or  spherically  corrected. 

If  the  correction  were  not  quite  suffi-  FIG.  168.  SPHERICAL  ABER- 
cient  so  that  the  border  rays  still  came  RATION- IN  LENSES. 

to  a  focus  slightly  nearer  the  lens  than  Axis  The  principal  optic 
the  middle  rays,  the  combination  would  7 '  2  3  Ray  x  at  the  ^e 

be  under-corrected.     If  the  concave  lens      comes  to  a  focus  at /i;  ray 

Al      i        ,  -  Al  2  at  /2,  and  ray  3  at  fa,  that 

were  too  strong,  the  border  rays  of  the      is>  the  nearer  the  optic  axis 

convex  lens  would  come  to  a  focus  farther  tne  longer  the  focus,  and  the 
r  ,,  ,  ,,  .,  . j,,  ,  nearer  the  edge  of  the  lens 

trom  the  lens  than  the  middle  rays,  and      the  shorter  the  focus. 

the  combination  would  be  said  to  be  over- 
corrected.  Sometimes  under-correction  or  over-correction  is  designed 
to  compensate  for  parts  of  the  optical  apparatus  which  the  rays  will 
meet  later,  or  for  aberrations  produced  before  the  light  reaches  the 
objective.  The  common  and  almost  universal  example  is  the  spher- 
ical aberration  introduced  by  the  cover-glass  over  the  specimen 
(fig.  169). 

§  460.  Cover-glass  correction.  —  By  referring  to  fig.  169  it  will 
be  seen  that  the  effect  of  the  cover-glass  is  precisely  like  the 
spherical  aberration  due  to  the  unequal  refraction  of  the  different 
zones  of  a  convex  lens;  that  is,  the  border  rays  are  more  bent  than 
those  nearer  the  axis,  as  the  obliquity  of  the  rays  is  greater 
(§446). 

Now  to  overcome  this  there  must  be  introduced  into  the  objective 
an  under-correction  just  sufficient  to  balance  the  effect  of  the  cover- 


286         TUBE-LENGTH    AND    COVER-GLASS    THICKNESS        [Cn  IX. 

glass.  If  the  lenses  are  fixed  in  position  in  the  objective  it  will 
be  evident  that  one  must  select  a  cover-glass  which  is  of  the  exact 
thickness  to  satisfy  the  correction  of  the  objective.  The  makers  of 
objectives  are  now  very  precise  in  stating  exactly  how  thick  the 
covers  should  be  for  their  objectives,  and  it  is  the  part  of  wisdom 
to  pay  heed  to  their  statements  if  one  hopes  to  get  the  best  re- 
sults. 

If  one's  objectives  are  adjustable  (§  134-135),  it  is  possible  to  so 
arrange  the  combinations  that  quite  a  range  of  cover-glass  thickness 


Slide 


I 


FIG.  169.   SPHERICAL  ABERRATION  INTRODUCED  BY  THE  COVER-GLASS. 

Axis  The  principal  optic  axis  extending  through  the  condenser  and  up  through 
the  object  and  microscope. 

Slide  The  glass  slide  on  which  the  object  is  mounted. 

Object   The  object  to  be  studied;  it  is  mounted  on  the  slide. 

Balsam  The  medium  in  which  the  object  is  mounted.  It  has  practically  the 
same  refractive  index  as  the  cover. 

Cover-glass  The  thin  glass  plate  over  the  object. 

i  2  3  The  light  rays  extending  obliquely  upward  from  the  object. 

3  2  i  Light  rays  traced  backward  to  their  apparent  origin,  the  most  oblique 
ray  (j)  being  most  bent,  thus  rendering  its  origin  apparently  highest. 

r  r  r   Points  of  refraction  of  the  three  oblique  rays. 

or  mounting  medium  thickness  can  be  used  and  still  get  the 'best 
optical  effect  by  balancing  the  aberrations  (§462). 

§  461.  Tube-length.  —  The  length  of  the  tube  on  the  microscope 
must  be  made  of  the  standard  for  which  the  objective  used  was  cor- 
rected or  aberrations  will  appear. 

If  the  tube  is  shorter  than  the  objective  was  corrected  for,  the 


CH.  IX] 


TUBE-LENGTH    OF    THE'  MICROSCOPE 


287 


effect  is  the  same  as  thinning  the  cover-glass.  That  is,  it  introduces 
under-correction.  This  makes  it  possible  to  compensate  for  too 
thick  a  cover  by  shortening  the  tube  (§  135,  462). 

If  the  tube  is  made  longer  than  the  standard  it  has  the  same  effect 
as  using  too  thick  a  cover-glass.  It  therefore  introduces  over-correc- 
tion, and  if  a  cover  too  thin  has  been  used  it  may  be  compensated 
for  by  lengthening  the  tube. 

When  homogeneous  immersion  liquid  is  used  one  does  not  have  to 
trouble  about  the  exact  thickness,  but  care  must  be  taken  not  to 
use  so  thick  a  cover  that  the  working 
distance  will  be  too  short  (§  76). 

By  consulting  the  catalogues  of  micro- 
scope manufacturers  one  can  find  for 
what  tube-length  and  thickness  of  cover- 
glass  their  unadjustable  objectives  are 
corrected.  For  example,  in  the  1914 
editions  oi  the  catalogues  of  the  Bausch 
&  Lomb  Optical  Company  of  Rochester, 
and  of  the  Spencer  Lens  Company  of 
Buffalo,  it  is  stated  that  the  tube-length 
is  1 60  millimeters  and  as  shown  in  the 
accompanying  figure  (fig.  170),  it  in- 
cludes the  parts  from  the  upper  end  of 
the  draw-tube  to  the  nut  into  which  the 
objective  is  screwed. 

The  cover-glass  thickness  is  given  as 
o.i  8  millimeter,  and  the  user  is  warned 

that  for  the  higher  powers  a  variation  in  thickness  from  this  standard 
of  0.03  or  0.04  mm.  would  deteriorate  markedly  the  perfection  of  the 
image.  The  statement  is  furthermore  made  that  with  the  homo- 
geneous immersions  no  harm  would  result,  but  on  the  other  hand 
great  care  must  be  exercised  there  to  use  the  correct  tube-length  or 
aberrations  will  be  introduced. 


CONDENSER 


FIG.  170.  THE  MICROSCOPE 
SHOWING  TUBE-LENGTH. 


288 


CORRECTION  OF  ABERRATIONS 


[CH.  IX 


§  462.     Table    showing    cause    of     spherical    aberration    in    the 
microscope  and  means  of  correction.  — 


Under-correction  produced  by: 
i.   Too  weak  a  concave  element  in  the 
objective.  * 
2.   Too  close  an  approximation  of  the 
lenses  of  the  objective. 
3.   Too  short  a  tube,  that  is  the  ocular 
and  objective  are  too  close  to- 
gether. 
4.   Use  of  too  thin  a  cover-glass. 

Over-correction  produced  by: 
la.   Too  strong  a  concave  element  in 
the  objective. 
2a.   Too   great    a    separation   of    the 
lenses  of  the  objective. 
3a.   Too  long  a  tube,  that  is  the  ocular 
and  objective  are  too  far  apart. 

4a.   Use  of  too  thick  a  cover-glass. 

Any  defect  can  be  neutralized  by  applying  the  right  amount  of 
what  would  produce  the  opposite  condition.  For  example,  the  over- 
correction  produced  by  too  thick  a  cover-glass  can  be  corrected  by: 
(4)  Using  a  thinner  cover-glass;  (3)  Shortening  the  tube;  (2)  Putting 
the  lenses  of  the  objective  closer  together;  (i)  using  a  weaker  con- 
cave element  in  the  objective. 

If  there  is  under-correction  from  too  short  a  tube  it  can  be  neutral- 
ized by:  (3a)  lengthening  the  tube;  (4a)  using  a  thicker  cover-glass; 
(2a)  separation  of  the  lenses  of  the  objective;  (la)  using  a  stronger 
concave  element  in  the  objective.  And  similarly  with  under-correction 
or  over-correction  from  any  cause;  opposites  neutralize. 

§  463.  Chromatic  aberration.  —  Spherical  aberration  which  has 
just  been  discussed  is  present  in  lenses  even  when  the  light  is  of  one 
wave  length;  chromatic  aberration,  on  the  other  hand,  appears  in 
addition  when  composite  light  traverses  a  lens.  This  is  because 
every  wave  length  of  necessity  is  differently  refracted;  the  shortest 
waves  most,  the  longest  waves  least.  If  then  a  single  beam  of  white 
light  traverses  a  lens,  the  different  wave  lengths  will  be  refracted  differ- 
ently and  the  blue-violet  waves  made  to  cross  the  axis  first,  the  red 
waves  last.  There  will  be  then  a  series  of  colored  foci  extending  along 
the  axis,  as  shown  in  fig.  171.  Every  simple  lens,  then,  whose  aperture 
is  filled  with  composite  light  will  show  both  spherical  and  chromatic 
aberration,  and  the  greater  the  aperture  and  the  shorter  me  focus 
the  more  pronounced  will  be  both  forms  of  aberration.  In  order  that 
perfect  images  may  be  produced,  both  aberrations  must  be  eliminated. 


CH.  IX] 


CORRECTION  OF  ABERRATIONS 


289 


Fortunately  the  visible  spectrum  does  not  include  a  greater  range 
of  wave  lengths  (fig.  151),  and  if  it  were  markedly  less  the  optician 
would  find  his  task  greatly  lightened.  As  shown  in  fig.  139,  the  bright- 
est region  of  the  spectrum  to  the  eye  is  really  limited,  and  the  old 
opticians  made  good  instruments  for  visual  purposes  by  overcoming 
the  aberrations  in  large  part  in  this  very  limited  region;  but  with  the 
requirements  of  photography  and 
for  the  most  complete  visual  study 
of  the  phenomena  and  objects  of 
nature  by  means  of  optical  in- 
struments greater  and  still  greater 
demands  were  made  for  optical 
instruments  including  at  least  the 
whole  visible  spectrum,  and  for  some 
purposes  extending  into  the  infra- 
red and  the  ultra-violet. 

§  464.  Correction  of  the  aberra- 
tions of  lenses.  —  From  the  very 
law  of  refraction  bound  up  with 
the  different  wave  lengths  of  visible 
light  it  would  seem  impossible  to 
obtain  the  refraction  necessary  to 
produce  images  (fig.  163-166)  with- 
out at  the  same  time  dividing  the 
light  up  into  its  colors.  If  the  re- 
fraction of  each  wave  length  were 
.  exact  proportion  to  its  length,  as 
with  a  diffraction  grating  it  would  be  impossible  to  produce  achro- 
matic images.  Newton  thought  the  refraction  was  always  as  with  a 
grating,  and  he  explained  the  satisfactory  images  produced  by  lenses 
on  the  ground  that  the  narrow  part  of  the  spectrum  most  brilliant  to 
the  eye  overwhelmed  the  dimmer  parts  so  that  the  colored  images  on 
both  sides  of  the  visual  image  were  ignored. 

If  one  compares,  however,  the  spectrum  produced  by  the  diffraction 
grating  (fig.  146)  with  that  produced  by  a  glass  prism  (fig.  147)  it 
will  be  seen  that  the  refraction  of  the  different  wave  lengths  (disper- 


FIG.  171.    CHROMATIC  ABERRATION 
WITH  COMPOSITE  LIGHT. 

White  light  A  beam  of  white  light 
composed  of  all  the  colors  meeting  a 
lens  and  the  different  wave  lengths 
being  differently  refracted  breaks  the 
composite  light  up  into  its  constitu- 
ent colors. 

Red  Blue  The  long  waved  red 
light  is  less  refracted  than  the  shorter 
waved  blue  light.  After  crossing  at 
the  foci  the  blue  light  is  on  the  outside 
of  the  diverging  cone. 

fb,  fr  The  focus  of  the  blue  light 
(fb}  nearer  the  lens  than  the  focus  of 
the  red  light  (/;). 

Axis    The  optic  axis  of  the  lens. 

The  dispersion  or  separation  into 
colors  differs  with  different  transpar- 
ent substances,  and  is  not  in  propor- 
tion to  the  mean  refraction. 


290          CORRECTION  OF  ABERRATIONS          [CH.  IX 

sion)  differs  very  markedly  in  the  two  cases,  although  the  total  length 
of  the  spectrum  is  the  same  in  both. 

The  red  is  much  contracted  and  the  blue  expanded  with  the  glass 
prism.  One  can  then  have  what  might  be  called  a  mean  refraction 
with  the  glass  prism,  the  refraction  of  the  individual  groups  of  wave 
lengths  not  being  in  proportion  to  the  lengths.  Now  it  is  from  this 
irregularity  of  the  refraction  in  different  parts  of  the  spectrum,  and 
because  the  irregularity  differs  with  different  transparent  substances, 


1  2 

FIG.  172.    ACHROMATISM  BY  COMBINING  DIFFERENT  KINDS  OF  GLASS. 

(1)  White  light  (W)  traversing  two  equal  crown  glass  (CC)  prisms  with  their 
bases  opposite.     The  dispersion  into  a  spectrum  by  the  first  prism  is  overcome  by 
the  second  prism  and  the  light  is  recombined  into  a  white  beam  (W1),  which  is 
displaced  as  if  it  had  traversed  a  piece  of  plane  glass. 

Red  Blue    The  red  and  the  blue  edges  of  the  spectrum.     The  blue  is  more 
refracted  than  the  red. 

(2)  White  light  (W)  traversing  a  flint  glass  prism  (F)  and  being  dispersed  into 
the  spectral  colors.     The  spectrum  formed  by  the  flint  prism  is  recombined  by  the 
crown  glass  prism  (C),  but  the  emerging  ray  of  white  light  (W2)  is  refracted  mark- 
edly toward  the  base  of  the  crown  glass  prism,  showing  the  possibility  of  an  achro- 
matic image.     The  arrows  show  the  direction  in  which  the  light  is  extending. 

that  it  is  possible  to  have  the  refraction  necessary  to  produce  images 
without  having  the  light  dispersed  into  colors  at  the  same  time.  This 
is  shown  in  fig.  172,  2,  where  a  smaller  prism  of  flint  glass  produces 
the  same  amount  of  dispersion  as  a  larger  prism  of  crown  glass.  If 
these  prisms  are  with  their  edges  opposite,  the  spectrum  produced 
by  the  flint  glass  will  be  brought  together  by  the  crown  glass  and 
white  light  will  result,  but  as  the  mean  refraction  of  the  larger  crown 
glass  prism  is  greater  than  that  of  the  flint  glass  prism,  the  ray  of 
white  light  will  not  extend  parallel  with  the  original  direction,  but 
be  bent  toward  the  base  of  the  crown  glass  prism.  As  a  lens  may  be 
considered  an  infinite  number  of  prisms  combined  it  becomes  intelli- 


CH.  IX]      CORRECTION  OF  APOCHROMATIC  OBJECTIVES  291 

gible  from  this  how  it  is  possible  to  -produce  colorless  images  by  com- 
bining flint-glass  concave  and  crown-glass  convex  lenses;  or  other 
pairs  of  lenses  where  the  dispersion  and  refraction  give  comparable 
results. 

In  making  the  color  corrections  for  the  lenses,  the  spherical  cor- 
rections were  also  made;  the  extent  of  both  corrections  attained  up 
to  the  present  is  discussed  below. 

§  465.   Corrections  in  Achromatic  and  Apochromatic  objectives.  - 
(i)   Spherical  aberration.     In  achromatic  objectives   the  spherical 


FIG.  173.     ACHROMATIC  COMBINATIONS  OF  CROWN  AND  FLINT  GLASS  LENSES. 
(From  Lewis  Wright's  Optical  Projection). 

C  C  C  C  C  C    Thin  edge  or  converging  crown  glass  lenses. 

F  F  F  F  F  F  Thick  edge  or  diverging  flint  glass  lenses.  The  flint  glass  over- 
comes the  dispersion  without  overcoming  the  mean  refraction,  hence  all  these 
combinations  are  converging. 

aberration  is  corrected  for  one  color  only;  in  apochromatic  objectives 
for  two  colors.  (2)  Chromatic  aberration.  In  achromatic  objectives 
correction  is  made  for  two  colors;  in  apochromats  for  three  colors. 

In  the  apochromats  it  was  found  impossible  to  make  the  high 
corrections  necessary  even  with  all  the  new  glasses  made  available 
by  the  Jena  glass  works;  but  with  the  new  forms  of  glass  and  a  natural 
mineral,  fluorspar,  fluorite,  calcium  fluoride,  with  its  very  low  index  of 
refraction  and  small  dispersion,  it  was  found  possible  to  make  the 
fundamental  advance  in  microscope  objectives  represented  by  the 
apochromatic  objectives. 

The  possibility  of  bringing  three  colors  to  one  focus  makes  the 
apochromatic  objectives  especially  valuable  for  photography.  The 


29 2  CORRECTION  OF  APOCHROMATIC  OBJECTIVES      LCn.  IX 

visual  and  actinic  foci  are  coincident,  and  if  the  apparatus  is  well 
constructed  there  is  never  any  difficulty  in  getting  sharp  pictures,  for 
the  photographic  image  is  sharpest  when  it  appears  sharpest  to  the 
normal  eye. 


FIG.  174.    POSITIVE  COMPENSATION  OCULAR. 
(From  Spitta,  p.  no). 

C  F  C  The  field-lens  is  composed  of  two  double  convex  crown  lenses  and  one 
double  concave  flint  glass  lens. 

C  The  eye-lens  is  of  crown  glass,  and  is  separated  from  the  field  combination 
the  right  distance  to  give  the  necessary  excess  magnification  of  the  red  image  to 
make  it  balance  the  blue  image  which  was  over  magnified  by  the  objective. 

Red  Blue  The  red  and  blue  rays  limiting  the  image.  It  is  seen  here  that  the 
rays  are  not  parallel  but  divergent,  as  they  extend  above  the  ocular.  When  pro- 
jected by  the  eye  to  the  virtual  image  the  rays  cross,  throwing  the  red  one  to  the 
outside,  thus  giving  a  larger  image  than  is  given  by  the  blue  ray,  and  the  orange 
haze  at  the  margin  of  the  field  when  looking  through  the  ocular  toward  the  window 
or  the  sky. 

§465a.  It  is  interesting  to  note  that  the  wonderful  optical  qualities  of  fluor- 
spar were  known  to  Sir  David  Brewster,  and  recommended  by  him  for  aid  in  achro- 
matization  (Brewster's  work  on  the  microscope,  1837,  p.  in);  and  before  1860  our 
own  Charles  A.  Spencer  used  fluorspar  in  one  of  the  combinations  of  his  objectives 
(Proc.  Acad.  Nat.  Sci.,  Phila.,  Vol.  LVI  (1904)^.475;  Trans.  Amer.  Micr.  Soc., 
1901,  p.  23) 

§  466.  Compensation  oculars.  —  As  the  front  lens  of  objectives  of 
high  power  (fig.  21,  B  C)  is  not  a  combination  but  a  single  lens, 


CH.  IX]         CORRECTION  OF  COMPENSATION  OCULARS 


293 


aberrations  are  inevitably  introduced  which  must  be  eliminated  by  a 
subsequent  part  of  the  optical  train.  The  most  striking  and  trouble- 
some defect  is  the  so-called  difference  of  chromatic  magnification; 


Ey«  Point  H 


|            Field 

8 

Len> 

f  ^-^\ 

H 

C 

FIG.  175.    HUYGENIAN  OCULAR  SHOWING  THE  ORDINARY  AND  THE  COMPENSATING 

ACTION. 

(From  Spitta,  p.  106). 

Ordinary  action.     (H). 

If  the  rays  are  traced  on  the  left  it  will  be  seen  that  the  field-lens  (C)  brings  the 
rays  to  a  focus  at  the  diaphragm  (D),  and  that  they  cross  and  pass  on  to  the  eye-lens 
slightly  divergent,  but  in  passing  through  the  eye-lens  (C),  the  red  and  blue  con- 
stituents are  made  parallel  to  each  other,  and  are  projected  into  the  field  of  vision 
in  close  parallel  (virtual)  bundles  and  hence  appear  achromatic. 

Compensating  action  (C). 

For  this  the  field-lens  is  of  flint  glass  (F),  and  the  eye-lens  of  crown  glass  (C). 
Or  the  eye-lens  may  be  an  over-corrected  combination.  The  end  result  is  the  same, 
viz.,  the  red  image  is  magnified  more  than  the  blue  image  by  the  ocular,  and  this 
balances  the  excess  magnification  of  the  blue  image  by  the  objective  and  in  the 
projected  (virtual)  image  the  red  is  on  the  outside,  producing  the  orange  haze 
at  the  margin  of  the  field  when  looking  through  the  ocular,  toward  a  window,  or 
the  sky. 

that  is,  the  differently  colored  constituent  images  forming  the  final 
image  are  of  different  magnitudes,  the  blue  one  being  larger  than  the 
red  one.  This  defect  is  more  easily  corrected  in  the  ocular  than  in 
the  subsequent  combinations  of  the  objective.  The  ocular  is  then 


294  ANGULAR  AND  NUMERICAL  APERTURE  [CH.  IX 

constructed  to  give  a  red  image  sufficiently  large  to  bring  its  mag- 
nification up  to  that  of  the  blue  image,  and  hence  the  final  image  as 
seen  by  the  eye  is  correct.  The  low  power  apochromats  could  be 
corrected  for  this,  but  for  the  sake  of  using  the  same  oculars  on  all 
powers  the  defect  is  left  or  purposely  introduced  into  all  the  apochro- 
mats. It  will  be  seen  from  the  above  statement  that  for  projection 
or  for  photography  the  apochromats  cannot  be  used  satisfactorily 
without  the  ocular  to  complete  the  corrections  (see  fig.  174-175). 

The  over-correction  of  the  ocular  necessary  to  give  the  greater  mag- 
nification to  the  red  constituent  of  the  image  leads  to  the  position  of 
the  red  on  the  outside  of  the  projected  (virtual)  beam;  hence  in  looking 
through  a  compensation  ocular  toward  the  window  or  the  sky  an 
orange  haze  appears  around  the  margin.  As  the  ordinary  Huygcnian 
ocular  has  an  under-corrected  eye-lens  the  blue  constituent  will  be  on 
the  outside  of  the  projected  (virtual)  image  and  there  appears  a 
blue  haze  around  the  edge  of  the  field  (Spitta,  p.  112-113). 

ANGULAR  AND  NUMERICAL  APERTURE 

§  467.  Angular  aperture.  —  By  this  is  meant  the  angle  of  light 
which  passes  from  the  object  to  the  objective  and  becomes  effective 
in  producing  the  microscopic  image  (fig.  176).  It  has  been  known  for 
a  very  long  time  that  the  clearness  of  the  image,  other  things  being 
equal,  depends  upon  the  width  of  the  angle  of  light  coming  from  the 
object;  and  that  the  resolution  of  details  depended  very  largely  upon 
the  angular  aperture  of  the  objective.  The  difficulty  of  overcoming 
the  aberrations  also  became  greater  as  the  angle  was  increased;  and 
it  was  the  triumph  of  the  early  American  opticians,  Spencer  and 
Tolles,  that  they  were  able  to  make  the  corrections  for  high  powers 
with  very  large  angular  aperture. 

§  468.  Numerical  aperture.  —  With  the  introduction  of  immersion 
systems  into  modern  microscopy,  it  was  seen  and  pointed  out  with 
great  distinctness  by  Spencer  and  Tolles  that  the  aperture  of  such 
immersion  objectives  might  exceed  180°  of  light  in  air.  For  the 
average  microscopist,  however,  this  seemed  an  impossibility.  By 
referring  to  fig.  158  to  160  the  matter  becomes  very  easily  intelligible, 


CH.  IX] 


ANGULAR  AND  NUMERICAL  APERTURE 


295 


for  it  is  seen  that  light  in  water  in  passing  into  air  spreads  out  so  that 

an  angle  in  water  of  48°  45'  on  each  side  of  the  normal  (97°  30')  spreads 

out  into  an  angle  of  180°  in  air;  therefore  light  at  an  angle  of  97°  36' 

in  water  is  equal  to  180°  in  air,  and  if  the  water  immersion  objective 

receives  and  transmits  for  the  formation  of  the  image  an  angle  of 

light  in  the  water  greater  than  97°  30'  its  angle 

is  greater  than  an  air  angle  of  180°.    In  the  same 

way  with  homogeneous  immersion.     The  critical 

angle  for  glass  to  air  is  41°  on  each  side  of  the 

normal,  and  a  total  angle  of  82°  in  the  glass 

would  spread  out  to  form  the  whole  180°  in  the 

air.    Therefore,  if  with  homogeneous  immersion 

objectives  an  angle  above  82°  is  transmitted  by 

the  objective  for  the  formation  of  the  image,  the 

angle  is  so  much  greater  than  180°  in  air. 

The  confusion  was  reduced  to  order  by  Abbe, 
to  whom  makers  and  users  of  optical  instruments 
owe  so  many  debts.  He  applied  the  simple 
laws  of  trigonometry,  using  the  sine  function  of 
the  angle,  and  taking  into  consideration  the 
medium  of  the  lowest  refractive  index  between 
the  object  and  the  objective.  If  it  were  air,  unity 
was  taken,  if  water  the  index  of  water  —  i-33; 
if  glass,  1.52;  and  if  any  other  immersion  fluid, 
the  refractive  index  of  that  fluid.  By  thus  con- 
sidering the  index  of  refraction  of  the  medium 
immediately  in  front  of  the  objective,  it  became 
possible  to  make  comparisons  which  were  rigidly  exact,  and  expressed 
in  terms  which  did  not  seem  impossibilities  like  an  angle  in  excess  of 
1 80°  to  enter  a  flat  surface. 

The  nomenclature  introduced  by  him  and  now  universally  em- 
ployed is  Numerical  Aperture,  and  includes  in  its  significance  both 
the  angle  of  the  light  and  the  index  of  refraction  of  the  medium  from 
which  the  light  passes  into  the  objective.  The  formula  is  N.A.  = 
n  sin  u,  in  which  n  is  the  index  of  refraction  of  the  air  for  dry,  the 
water  for  water  immersion  and  the  cedar  oil  for  homogeneous  im- 


FIG.  176.  ANGULAR 
APERTURE  OF  AN  OB- 
JECTIVE. 

Axis,  The  princi- 
pal optic  axis  of  the 
objective. 

B  The  object  just 
outside  the  principal 
focus. 

ADC  Diameter  of 
the  front  of  the  objec- 
tive and  base  of  the 
angle  of  aperture. 

A  D  B  Half  the 
angle  of  aperture  00; 
AD  representing  the 
sine  of  u  (see  §  468). 


296 


ANGULAR  AND  NUMERICAL  APERTURE 


[CH.  IX 


mersion;  and  u,  is  the  sine  of  half  the  angle  of  the  light  entering  the 
microscope  objective,  no  matter  what  medium  is  between  the  object 
and  objective. 

As  there  are  three  factors  in  this  formula,  if  one  knows  any  two  of 
them  the  third  is  readily  found: 


§  469.  Table  of  a  Group  of  Objectives  with  their  Numerical  Aperture  with  Method  of 
Obtaining  It.     For  a  Table  of  Natural  sines,  see  third  page  of  cover. 


Objective 

|h 

Natural  Sine 
of  half  the  angular 
aperture 
(sin  u). 

Index  of 
Refraction 
of  the  medi- 
um in  front 
of  the  objec- 
tive («) 

Numerical  Aperture 
(N.  A.)  =  n  sin  u 

25  mm. 
Dry. 

20° 

Sin  —  -  =  0.1736 

n  =  i 

N.A. 

=      IX  0.1736  =  0.173 

25  mm. 
Dry. 

40° 

Sin      -  =  0.3420 

n.=  i 

N.A. 

i  X  0.3420=0.342 

125  mm. 
Dry. 

42° 

Sin  —  =  0.3584 

n  =  i 

N.A. 

=      IX  0.3583  =  0.358 

12^  mm. 
Dry. 

100° 

.      100° 

Sin  =  0.7660 

n  =  i 

N.A. 

=      I  X  0.7660  =  0.766 

6  mm. 
Dry. 

75° 

•7  C° 

Sin  —  =  0.6087 

n  =  i 

N.A. 

=      IX  0.6087  =  0.609 

6  mm. 
Dry. 

136° 

Sin-—  =  0.9272 

;/  =  i 

N.A. 

=      I  X  0.9272  =  0.927 

3  mm. 
Dry. 

H5° 

Sin  --  —  =  0.8434 

n  =  i 

N.A. 

=      I  X  0.8434  =  0.843 

3  mm. 
Dry. 

163° 

Sin  —  —  =  0.9890 

n  =  i 

N.A. 

=      IX  0.9890  =  0.989 

2  mm. 
Water 
Immersion 

96°!  2' 

.    96°i  2' 
Sin               —  o  *T4-i'i 

n  =  1.33 

N.A. 

=  1.33  X  0.7443  =  0.99 

2  mm. 
Homogeneous 
Immersion 

no°38' 

.    no°38' 
Sin  *—  =  0.8223 

n  =  1.52 

N.A. 

=  1.52  X  0.8223  =  1.25 

2  mm. 
Homogeneous 
Immersion 

i34°io' 

i34°io' 

n  =  1.52 

N.A. 

=  1.52  X  0.9210  =  1.40 

Sin               —  0.921  1 

CH.  IX]  FINDING  THE  NUMERICAL  APERTURE  297 

§  470.  Significance  of  numerical  aperture.  —  It  is  now  universally 
agreed  that,  the  corrections  in  chromatic  and  spherical  aberration 
being  the  same,  the  power  to  define  minute  details  depends  directly 
on  the  numerical  aperture;  the  greater  the  numerical  aperture  the 
greater  the  resolution  (see  also  §  475-476). 

§  471.  Why  a  homogeneous  immersion  condenser  is  required.  — 
If  the  definition  of  minute  details  requires  adequate  numerical  aper- 
ture it  is  evident  that  it  is  of  fundamental  importance  that  the  sub- 
stage  condenser  be  able  to  supply  the  light  at  the  adequate  aperture. 
Assuming  that  the  substage  condenser  is  properly  constructed,  the 
question  is,  can  it  illuminate  the  object  with  the  proper  numerical 
aperture? 

By  referring  to  §  468,  and  to  figures  158-160,  it  is  evident  that  an 
object  mounted  on  a  glass  slide  and  separated  from  the  condenser  by  a 
stratum  of  air  can  get  light  from  the  condenser  only  up  to  the  critical 
angle,  that  is  41°,  on  each  side  of  the  normal,  or  a  total  of  82°,  cor- 
responding to  a  numerical  aperture  of  i.  The  objective  may  be  cap- 
able, however,  of  receiving  and  utilizing  a  numerical  aperture  of  1.40. 

If  now  the  condenser  also  has  a  numerical  aperture  of  i  .40  and  it  is 
connected  to  the  slide  by  means  of  homogeneous  immersion  liquid  the 
entire  aperture  will  illuminate  the  object  and  can  enter  the  homo- 
geneous immersion  objective. 

If  the  substage  condenser  is  connected  with  the  slide  by  means  of 
water,  then,  as  shown  in  fig.  160,  the  object  can  be  illuminated  with 
an  angle  of  61°  -f  61°  or  122°,  or  a*.numerical  aperture  of  n  sin  u;  in 
this  case  1.3^  x  0.875  =  I-***7-  If  the  greatest  possible  aperture  is 
required,  as  in  dark-ground  illumination  (§  125),  and  for  some  of  the 
most  exacting  work  with  photo-micrography  and  microscopic  study, 
the  condenser  should  be  connected  with  the  slide  by  homogeneous 
immersion  liquid. 

§  472.  Determination  of  the  aperture  of  objectives  with  an  aper- 
tometer.  —  Excellent  directions  for  using  the  Abbe  Apertometer  may 
be  found  in  the  Jour.  Roy.  Micr.  Soc.,  1878,  p.  19,  and  1880,  p. 
20;  in  Dippel,  Czapski  and  Spitta,  Chapter  XIV.  The  following 
directions  are  but  slightly  modified  from  Carpenter-Dallinger,  pp. 
394-396.  The  Abbe  apertometer  involves  the  same  principle  as  that 


298 


FINDING  THE  NUMERICAL  APERTURE 


[CH.  IX 


of  Tolles,  but  it  is  carried  out  in  a  simpler  manner;  it  is  shown  in 
fig.  177.  As  seen  by  this  figure  it  consists  of  a  semicircular  plate  of 
glass.  Along  the  straight  edge  or  chord  the  glass  is  beveled  at  45°, 
and  near  this  straight  edge  is  a  small,  perforated  circle,  the  perfora- 
tion being  in  the  center  of  the  circle.  To  use  the  apertometer  the 
microscope  is  placed  in  a  vertical  position,  and  the  perforated  circle 
is  put  under  the  microscope  and  accurately  focused.  The  circular 
edge  of  the  apertometer  is  turned  toward  a  window  or  plenty  of 
artificial  light  so  that  the  whole  edge  is  lighted.  When  the  objective 


FIG.  177.   ABBE  APERTOMETER. 

is  carefully  focused  on  the  perforated  circle  the  draw -tube  is  removed 
and  in  its  lower  end  is  inserted  the  special  objective  which  accom- 
panies the  apertometer.  This  objective  and  the  ocular  form  a  low 
power  compound  microscope,  and  with  it  the  back  lens  of  the  objective, 
whose  aperture  is  to  be  measured,  is  observed.  The  draw-tube  is 
inserted  and  lowered  until  the  back  lens  of  the  objective  is  in  focus, 
"In  the  image  of  the  back  lens  will  be  seen  stretched  across,  as  it  were, 
the  image  of  the  circular  part  of  the  apertometer.  It  will  appear  as  a 
bright  band,  because  the  light  which  enters  normally  at  the  surface  is 
reflected  by  the  bevel  part  of  the  chord  in  a  vertical  direction  so  that 
in  reality  a  fan  of  180°  in  air  is  formed.  There  are  two  sliding 
screens  seen  on  either  side  of  the  apertometer;  they  slide  on  the  verti- 
cal circular  portion  of  the  instrument.  The  images  of  these  screens 
can  be  seen  in  the  image  of  the  bright  band.  These  screens  should  now 
be  moved  so  that  their  edges  just  touch  the  periphery  of  the  back  lens. 
They  act,  as  it  were,  as  a  diaphragm  to  cut  the  fan  and  reduce  it,  so 
that  its  angle  just  equals  the  aperture  of  the  objective  and  no  more." 


CH.  IX]  TESTING  HOMOGENEOUS  LIQUID  299 

"This  angle  is  now  determined  by  the  arc  of  glass  between  the  screens; 
thus  we  get  an  angle  in  glass  the  exact  equivalent  of  the  aperture  of 
the  objective.  As  the  numerical  apertures  of  these  arcs  are  engraved 
on  the  apertometer  they  can  be  read  off  by  inspection.  Nevertheless 
a  difficulty  is  experienced,  from  the  fact  that  it  is  not  easy  to  deter- 
mine the  exact  point  at  which  the  edge  of  the  screen  touches  the 
periphery  of  the  back  lens,  or  as  we  prefer  to  designate  it,  the  limit  of 
aperture,  for  curious  as  the  expression  may  appear  we  have  found  at 
times  that  the  back  lens  of  the  objective  is  larger  than  the  aperture 
of  the  objective  requires.  In  that  case  the  edges  of  the  screen  refuse 
to  touch  the  periphery." 

In  determining  the  aperture  of  homogeneous  immersion  objectives 
the  proper  immersion  fluid  should  be  used  as  in  ordinary  observation. 
So,  also,  with  glycerin  or  water  immersion  objectives. 

§  473.  Testing  Homogeneous  Immersion  Liquid.  —  In  order  that 
one  may  realize  the  full  benefit  of  the  homogeneous  immersion  prin- 
ciple it  is  necessary  that  the  homogeneous  immersion  liquid  shall  be 
truly  homogeneous.  In  order  that  the  ordinary  worker  may  be  able 
to  test  the  liquid  used  by  him,  Professor  Hamilton  L.  Smith  devised 
a  tester  composed  of  a  slip  of  glass  in  which  was  ground  accurately  a 
small  concavity  and  another  perfectly  plain  slip  to  act  as  cover. 
(See  Proc.  Amer.  Micr.  Soc.,  1885,  p.  83.)  It  is  readily  seen  that  this 
concavity,  if  filled  with  air  or  any  liquid  of  less  refractive  index  than 
glass,  acts  as  a  concave  or  dispersing  lens.  If  filled  with  a  liquid  of 
greater  refractive  index  than  glass,  the  concavity  acts  like  a  convex 
lens,  but  if  filled  with  a  liquid  of  the  same  refractive  index  as  glass, 
that  is,  liquid  optically  homogeneous  with  glass,  then  there  is  no  effect 
whatever. 

In  using  this  tester  the  liquid  is  placed  in  the  concavity  and  the 
cover  put  on.  This  is  best  applied  by  sliding  it  over  the  glass  with  the 
concavity.  A  small  amount  of  the  liquid  will  run  between  the  two 
slips,  making  optical  contact  on  both  surfaces.  One  should  be  careful 
not  to  include  air  bubbles  in  the  concavity.  The  surfaces  of  the  glass 
are  carefully  wiped  so  that  the  image  will  not  be  obscured.  An  adapter 
with  society  screw  is  put  on  the  microscope  and  the  objective  is  attached 
to  its  lower  end.  In  this  adapter  a  slot  is  cut  out  of  the  right  width 


300  TESTING  HOMOGENEOUS  LIQUID  [CH.  IX 

and  depth  to  receive  the  tester  which  is  just  above  the  objective.  As 
object  it  is  well  to  employ  a  stage  micrometer  and  to  measure  carefully 
the  diameter  of  the  field  without  the  tester,  then  with  the  tester  far 
enough  inserted  to  permit  of  the  passage  of  rays  through  the  glass 
but  not  through  the  concavity,  and  finally  the  concavity  is  brought 
directly  over  the  back  lens  of  the  objective.  This  can  be  easily  deter- 
mined by  removing  the  ocular  and  looking  down  the  tube. 

Following  Professor  Smith's  directions  it  is  a  good  plan  to  mark  in 
some  way  the  exact  position  of  the  tube  of  the  microscope  when  the 
micrometer  is  in  focus  without  the  tester,  then  with  the  tester  pushed 
in  just  far  enough  to  allow  the  light  to  pass  through  the  plane  glass, 
and  finally  when  the  light  traverses  the  concavity.  The  size  of  the 
field  should  be  noted  also  in  the  three  conditions  (§  47-49) . 

It  is  seen  by  glancing  at  the  following  table  that  whenever  the 
liquid  in  the  tester  is  of  lower  index  than  glass,  the  concavity  with  the 
liquid  acts  as  a  concave  lens,  or  in  other  words  like  an  amplifier 
(§  236a),  and  the  field  is  smaller  than  when  no  tester  is  used.  It  is 
also  seen  that  as  the  liquid  in  the  concavity  approaches  the  glass  in 
refractive  index,  the  field  approaches  the  size  when  no  tester  is 
present.  It  is  also  plainly  shown  by  the  table  that  the  greater  the 
difference  in  refractive  index  of  the  substance  in  the  concavity  and 
the  glass,  the  more  must  the  tube  of  the  microscope  be  raised  to 
restore  the  focus. 

If  a  substance  of  greater  refraction  than  glass  is  used  in  the  tester 
the  field  is  larger,  i.  e.,  the  magnification  less,  and  one  would  have  to 
turn  the  tube  down  instead  of  up  to  restore  the  focus. 

The  table  given  below  indicates  the  changes  when  using  a  tester 
prepared  by  the  Gundlach  Optical  Co.,  and  used  with  a  16  mm. 
apochromatic  objective  of  Zeiss,  X  4  compensation  ocular,  achro- 
matic condenser,  i.oo  N.  A.  (fig.  40): 

§  474.  Diffracted  light  in  microscopy.  —  As  most  microscopic  ob- 
servation depends  upon  directed  light  from  some  source  like  the  sun 
or  a  lamp  sent  to  and  through  the  object  by  a  mirror  only  or  by  the 
aid  of  a  condenser  or  a  mirror  and  condenser,  the  phenomena  of  dif- 
fraction are  present.  It  is  evident  that  if  the  objects  observed  were 
self-luminous  the  conditions  would  be  different  from  those  existing 


CH.  IX] 


DIFFRACTED   LIGHT  IN   MICROSCOPY 


3'oi 


Tester  and  Liquid  in  the  Concavity 

Size  of  the 
Field 

Elevation  of  the  Tube 
necessary  to 
Restore  the  Focus 

No  tester  used  
Whole  thickness  of  the  tester  at  one  end,  not 
over  the  cavity  
Tester  with  water  

1.825  mm..  .  . 

1.85  mm  
1.075  mm.  .  .  . 

Standard  position 

No  change  of  focus. 
Tube  raised  ^j  mm. 

Tester  with  95  %  alcohol 

i  15  mm 

3  mm 

Tester  with  kerosene  . 

i  4  mm 

2  mm 

Tester  with  Gundlach  Opt.  Co.'s  horn,  liquid. 
Bausch  &  Lomb  Opt.  Co.'s  horn,  liquid  
Leitz'  horn,  liquid  
Zeiss'  horn  liquid 

1.825  mm..  .  . 
1.825  mm..  .  . 
1.825  mm..  .  . 
i  825  mm 

iVo  mm- 
T2o°<y  mm. 
V&mm. 

TffO 

when  the  object  must  be  viewed  with  direct  light  from  some  outside 
source. 

In  traversing  small  orifices  or  slits  and  objects  with  minute  details 
the  spreading  out  of  diffracted  light  is  a  necessary  accompaniment. 
The  diffracted  rays  are  shown  by  broken  lines  in  the  accompanying 
figures  from  Wright  (fig.  178-179).  As  seen  from  these  there  may  be 
two  systems  of  diffracted  rays,  one  from  the  object  and  another  from 
the  border  of  the  objective,  and  these  two  systems  of  diffracted  rays 
act  differently. 

The  role  played  by  the  diffracted  light  has  been  variously  inter- 
preted by  opticians.  By  Abbe  and  his  adherents  diffracted  light  is  of 
supreme  importance,  and  microscopic  vision  is  a  thing  by  itself  (sui 
generis)  and  not  to  be  interpreted  by  ordinary  geometric  optics. 
Certain  very  striking  experiments  have  been  devised  to  show  the  accu- 
racy of  this  hypothesis,  but  as  pointed  out  by  many,  the  ordinary  use 
of  the  microscope  never  involves  the  conditions  realized  in  those 
experiments. 

While  the  supreme  importance  ascribed  by  some  to  the  diffracted 
light  may  not  be  accepted,  no  one  will  deny  the  presence  of  diffraction 
phenomena  in  miscroscopic  vision.  If,  furthermore,  the  diffracted  rays 
are  brought  by  the  microscope  to  the  final  focus  with  the  undiffracted 
light  passing  from  the  object  through  the  microscope,  the  image  will 
be  conceivably  more  perfect  than  as  if  the  diffracted  rays  produce 
secondary  images,  or  mere  blur. 


302 


DIFFRACTED   LIGHT  IN  MICROSCOPY 


[CH.  IX 


FIG.  178-179.  DIFFRACTED  LIGHT  IN  MICROSCOPY. 
(From  Wright's  Principles). 

Fig.  178.  Object  (grating)  lighted  with  a  narrow  beam  (/)  from  the  condenser 
and  giving  off  diffracted  rays  which  are  brought  to  a  focus  with  the  dioptric  beam 
(I)  above  the  objective  in  part  (full  lines);  and  in  part  forming  diffracted  beams  on 
each  side  above  the  objective  (broken  lines).  These  diffracted  beams  not  brought 
to  the  same  focus  as  the  dioptric  beam  cause  imperfections  or  confusion  in  the 
image. 

Fig.  179.  Small  diaphragm  (C  D)  below  the  condenser  focused  on  the  grating, 
A  B,  and  from  this  point  the  diptric  beam  (solid  white)  and  diffracted  light  (broken 
lines)  extend  through  the  objective  and  finally  focus  at  B'  A'.  By  looking  at  the 
eye-point  with  a  magnifier  the  image  of  the  back  lens  shows  not  only  the  diaphragm 
image  (Df  C'),  but  secondary  images  of  the  same  (D'  C'  and  D"  C").  See  small 
figure  in  the  middle  also. 


CH.  IX]  APERTURE  AND  DEPTH  OF   FOCUS  30^ 

§  475.  Depth  of  focus  and  aperture.  —  It  is  known  to  all  workers 
with  the  microscope  that  with  objectives  of  low  aperture  it  is  possible 
to  change  the  focus  rather  markedly  up  or  down  without  seeming  to 
lose  in  sharpness,  while  with  objectives  of  great  aperture  a  sharp 
focus  is  almost  immediately  lost  in  focusing  up  or  down  beyond  a 
point.  The  reason  for  this  is  made  strikingly  evident  by  fig.  180 
(i,  2).  Let  /  be  the  most  perfect  focus,  if  one  turns  to  a  or  b  the 
appearance  is  almost  unchanged  in  the  low  apertured  objective  (2),, 
but  the  diffusion  circle  is  very  marked  in  the  high  apertured  objec- 
tive (i).  Furthermore,  the  brilliancy  of  the  image  must  be  markedly 
greater  with  the  larger  aperture  (Wright,  p.  77). 

§  476.  Aperture  and  the  effect  of  opacities.  —  Between  the  retina 
and  the  object  there  are  many  possibilities  of  opacities  in  the  image- 
producing  beam  of  light.  For  example,  the  eye  lashes,  particles  of 
dirt  in  the  tears  over  the  cornea,  besides  particles  on  the  glass  sur- 
faces. Figure  180  (3,  4,  5)  show  graphically  the  relative  obscuration 
which  must  result  with  the  same  opacity  in  beams  of  different  aper- 
ture. In  (3)  the  shadow  is  so  great  that  almost  the  entire  aperture  is 
obscured,  and  vision  made  difficult  or  impossible.  In  (4)  with  a 
larger  aperture  the  shadow  is  not  so  overwhelming,  and  in  (5)  with 
the  large  aperture  there  is  still  possibility  of  fairly  good  vision  in 
spite  of  the  shadow. 

It  is  believed  that  the  inevitable  narrowing  of  the  beam  in  high 
power  magnification  and  the  presence  of  opacities  in  the  eye  form 
the  bar  to  resolution,  and  that  if  the  apparatus  and  the  eye  could,  on 
the  one  hand,  be  free  from  opacities  to  throw  shadows  and  thus 
obscure  the  image,  or  on  the  other  hand  the  terminal  beam  could  be 
opened  up  to  make  the  aperture  greater,  the  eye  could  discriminate 
beyond  the  limits  heretofore  ascribed  to  it  (Wright,  Ch.  XVI). 

As  the  higher  the  power  of  the  ocular  the  smaller  is  the  eye-point 
(fig.  23-24),  it  is  evident  that  any  obscurities  have  a  greater  effect 
•with  the  high  ocular.  The  rule  to  use  as  low  an  ocular  as  possible  is  a 
good  one  to  follow  from  every  standpoint  (Wright,  p.  227). 


304 


APERTURE  AND  OPACITIES 


CCH.  IX 


FIG.  1 80.  EFFECT  OF   APERTURE   ON   DEFINITE   Focus   AND   ON  OVERCOMING 

OPACITIES. 

(From  Wright's  Principles  of  Microscopy,  p.  77). 

(1)  To  show  the  definiteness  of  the  focus  (/)  with  a  large  aperture.     Either 
above  or  below  this  is  a  large  diffusion  circle  (a  b]  due  to  the  size  of  the  section  of 
the  aperture. 

(2)  Indefiniteness  of  the  focus  due  to  the  fact  that  a  cross  section  of  the  aperture 
considerably  above  or  below  the  true  focus  (/),  gives  so  small  a  diffusion  circle  (a 
or  b) ,  that  it  can  hardly  be  distinguished  from  the  true  focus. 

(3)  Low  aperture  and  an  opacity  in  the  path  of  the  light.     It  is  so  large  relatively 
here  that  a  clear  image  would  be  impossible. 

(4)  The  same  opacity  in  a  larger  aperture. 

(5)  The  same  opacity  in  a  still  larger  aperture.     There  is  now  enough  of  the 
beam  outside  the  opacity  to  make  the  object  visible. 


CH.  IX]     MAGNIFICATION  OF  OBJECTIVES  AND  OCULARS          305 

• 
INDEPENDENT  MAGNIFICATION  OF  OBJECTIVES  AND  OCULARS 

§  477.   Independent   magnification   of   an   objective.  —  The   inde- 
pendent magnification  of  an  objective  is  like  that  of  a  simple  micro- 

Image 


Object 


FIG.  181.  REAL  IMAGE  SHOWING  THAT  THE  SIZE  DEPENDS  UPON  THE  .RELATIVE 
DISTANCE  OF  OBJECT  AND  IMAGE  FROM  THE  CENTER  OF  THE  LENS. 

Object    The  object  of  which  an  image  is  to  be  formed. 

cl    Center  of  the  lens. 

i  This  shows  that  the  object  is  i  unit  long  and  its  distance  from  the  center  of 
the  lens  is  also  i . 

1234  The  image  is  4  units  distant  from  the  center  of  the  lens,  and  the  image 
is  consequently  4  units  long. 


306          MAGNIFICATION  OF  OBJECTIVES  AND  OCULARS     [Cn.  IX 

scope  or  it  is  like  that  of  a  projection  microscope  when  the  objective 
alone  is  used  (fig.  181,  182).  As  pointed  out  in  §  236  it  is  necessary 
to  select  some  standard  distance  for  the  projection  of  the  real  or  of 


FIG.  182.  PROJECTED  VIRTUAL  IMAGE  OF  A  MAGNIFIER. 
(Simple  Microscope  or  Ocular). 

The  projection  distance  is  250  mm.  from  the  nodal  point  in  the  crystalline  lens 
of  the  eye. 

Axis    The  optic  axis  of  the  magnifier  and  of  the  eye. 

A1  B1    The  object  of  the  simple  microscope  or  real  image  of  the  objective. 

B2  Az    The  retinal  image  (inverted). 

A*  B3  The  projected  virtual  image.  It  is  erect  as  compared  with  the  object, 
but  inverted  as  compared  with  the  retinal  image. 

Cr.     Cornea  of  the  eye. 

R     Single  refractive  surface  of  the  schematic  eye.' 

L    Crystalline  lens  of  the  eye. 

the  virtual  image,  for  the  size  of  the  image  varies  directly  as  its  dis- 
tance from  the  center  of  the  lens  (fig.  181  for  real  and  182  for  virtual 
images;  in  the  latter  the  projection  distance  is  from  the  nodal  point 
in  the  eye  to  the  image).  The  image  distance  for  magnification  most 
commonly  employed  is  250  mm.  (§  236). 


CH.  IX]     MAGNIFICATION  OF  OBJECTIVES  AND  OCULARS          307 

To  find  the  magnification  of  any  objective  at  this  image  distance 
one  can  proceed  as  for  the  simple  microscope,  but  the  better  method 
is  by  the  use  of  the  ocular  micrometer.  Two  micrometers  of  known 
value  are  needed,  —  a  stage  micrometer  and  an  ocular  micrometer  in 
divisions  of  a  millimeter. 

(A)  Determination  of  the  magnification  with  a  Huygenian  ocular 
with  fixed  or  movable  scale  (fig.  91).    Remove  the  field-lens  and  focus 
the  ocular  micrometer  lines  by  raising  or  lowering  the  eye-lens.    Focus 
the  stage  micrometer  lines,  and  make  the  lines  of  the  two  micrometers 
parallel.    Make  the  lines  of  the  two  coincide.     Suppose  the  image  of 
0.2  mm.  on  the  stage  micrometer  covered  2mm.  on  the  ocular  mi- 
cometer,  the  magnification  in  this  case  would  be  2mm.  divided  by 
0.2  mm.  =  10.    One  could  also  use  the  filar  micrometer  as  directed 
in  §  243.    A  positive  ocular  has  the  advantage  that  nothing  needs  to 
be  changed  in  it. 

(B)  Effect  of  the  field -lens.    For  getting  the  independent  mag- 
nification of  the  objective  the  field-lens  of  the  Huygenian  ocular  must 
be  removed,  but  for  determining  the  magnification  of  the  objective 
when  used  with  the  field-lens  in  position  as  in  ordinary  observation, 
reinsert  the  field-lens  and  determine  the  magnification  of  the  com- 
bined objective  and  the  field-lens  exactly  as  directed  above.     One 
can  tell  in  this  way  also  how  much  the  magnification  of  the  ocular  is 
reduced  by  the  field-lens.    It  is  very  marked  (fig.  23-24,  183). 

(C)  Effect  of  tube-length.     The  effect  of  tube-length  on  the  magni- 
fication of  the  objective  is  discussed  in  §  236.  The  general  law  is  that 
with  a  given  lens  or  combination  the  more  distant  from  the  lens  the 
image  is  formed  the  greater  is  the  magnification;   therefore  in  every 
case  the  conditions  must  all  be  made  exactly  alike  if  the  results  are 
to  be  similar.    This  is  easily  proved  by  getting  the  magnification  of 
the  objective  on  the  ocular  micrometer  with  a  tube-length  of  160  mm. 
and  then  with  250  mm.    If  one  has  a  projection  microscope  the  dif- 
ference is  strikingly  shown  by  using  an  objective  alone  and  getting 
the  magnification  at  a  screen  distance  of  i  meter  and  then  at  2  meters. 
The  magnification  will  be  almost  exactly  twice  as  great  at  2  meters 
as  at  one  meter.    The  same  holds  for  the  projected  virtual  image,  as 
one  can  see  by  fig.  85  and  182. 


3o8 


MAGNIFICATION  OF  OBJECTIVES  AND  OCULARS    [Cn.  IX 


§  478.  Magnification  due  to  the  ocular.  —  To  find  this  experimen- 
tally use  a  positive  ocular  with  an  ocular  micrometer  or  a  filar  mi- 
crometer, or  remove  the  field-lens  of  the  Huygenian  ocular  in  which  is 
present  an  ocular  micrometer.  Make  the  tube-length  160  or  250  mm. 
Focus  the  stage  micrometer  on  the  ocular  micrometer  and  see  what 
the  objective  magnification  is.  For  example,  suppose  the  objective 
real  image  of  y1^  mm.  covers  2  mm.  on  the  ocular  micrometer,  the 


Ocular 


Objective 


FIG.  183.  DIAGRAM  SHOWING  THE  RAYS  AND  IMAGES  WHEN  BOTH  AN 
OBJECTIVE  AND  AN  OCULAR  ARE  USED. 

(From  Optic  Projection). 

Object    The  object  of  which  an  image  is  desired. 
Objective    The  optical  combination  forming  the  first  image. 
Ocular    Huygenian  ocular  projecting  the  screen  image. 
/  /     Field-lens  diminishing  the  real  image  of  the  objective  from  r'  /'  to  r  i. 
r  i     Real  image  produced  by  the  objective  and  the  field-lens. 
r'  i'    This  would  be  the  size  of  the  real  image  if  no  field-lens  were  used. 
e  I    Eye-lens  serving  to  project  the  real  image  (r  /)  to  the  screen. 
Screen  Image    The  real  image  on  the  screen  projected  by  the  eye-lens. 
Used  in  the  ordinary  way  with  the  eye  next  the  ocular  a  virtual  image  is  pro- 
jected (fig.  78,  182). 

magnification  in  that  case  is  20.  Now  use  the  Wollaston  camera 
lucida  and  project  the  virtual  image  250  mm.  and  get  the  magnifica- 
tion of  the  entire  microscope  as  directed  in  §  234.  Suppose  it  is  200 
diameters.  It  is  known  that  the  objective  magnifies  20  diameters 
and  to  get  200  diameters  there  must  be  a  second  or  ocular  magnifi- 
cation of  200  -f-  20  =  10.  That  is,  the  ocular  in  this  case  magnifies 
the  objective  real  images  10  diameters,  making  the  magnification  of 
the  microscope  as  a  whole  20  x  10  =  200. 

This  was  without  the  field-lens.    Put  the  field-lens  in  place  and  get 
the  magnification  of  the  entire  microscope  again.    It  will  be  markedly 


CH.  IX]  PAR-FOCAL  OCULARS  AND  OBJECTIVES  309 

less,  as  the  field-lens  makes  the  objective  image  smaller  (fig.  23-24, 
183).  In  the  case  in  hand  the  reduction  of  the  objective  image  was 
\ ,  so  that  the  real  image  of  the  objective  with  the  field-lens  in  place 
was  15  diameters,  and  of  the  whole  microscope  then  only  150  diameters, 
as  the  magnification  of  the  eye-lens  is  unchanged.  But  as  the  objec- 
tive is  not  changed  in  power  by  the  field-lens,  the  effect  of  the 
entire  ocular,  field-  and  eye-lens,  must  be  the  entire  magnification  of 
the  microscope  divided  by  the  power  of  the  objective,  which  is  20 
diameters.  150  -4-20  =  7.5.  In  this  case  then  the  ocular  as  a  whole 
magnifies  the  real  image  of  the  objective  (20)  7.5  times.  In  all  Huy- 
genian  oculars,  then,  the  field-lens  acts  as  a  reducing  lens,  the  eye- 
lens  as  a  magnifier,  and  this  is  true  whether  the  microscope  is  used  as 
in  ordinary  observation  (fig.  23-24)  or  for  projection  (fig.  183). 

§  479.  Magnification  of  drawings.  —  In  determining  the  magnifi- 
cation of  a  drawing  made  with  a  camera  lucida  or  with  projection- 
apparatus,  by  far  the  best  method  of  determining  it  is  to  remove  the 
specimen  and  put  in  its  place  a  stage  micrometer  and  project  the 
image  of  the  micrometer  upon  the  drawing  paper.  Make  a  few  lines 
of  the  micrometer  image  and  indicate  the  value  of  the  spaces  (fig. 
103),  then  at  any  time  one  can  determine  exactly  what  the  magnifi- 
cation is  (§  276). 

§  480.  Par-focal  Oculars.  —  By  this  is  meant  oculars  of  different 
power  in  which  the  microscope  remains  in  focus  on  changing  the  oculars. 

As  originally  constructed  the  microscope  had  to  be  focused  every 
time  the  oculars  were  changed.  Mr.  Edward  Pennock,  in  seeking  to 
overcome  this  inconvenience,  wrote  to  Professor  Abbe  for  advice  in 
1 88 1.  After  successfulfy  producing  oculars  of  different  powers  for 
the  Acme  microscopes  of  James  W.  Queen  &  Co.,  according  to  the 
directions  given  by  Professor  Abbe,  Mr.  Pennock,  as  editor  of  the 
Microscopical  Bulletin  and  Science  News,  published  in  Vol.  Ill,  1886, 
pp.  9-10,  the  following,  with  Professor  Abbe's  letter:  "Changing 
Eye-pieces  without  altering  Focus,"  etc.  "  Some  years  ago  the  writer, 
in  looking  up  certain  questions  in  connection  with  eye-pieces,  took 
occasion  to  write  to  Professor  Abbe,  and  his  reply,  kindly  given,  is  so 
clear  and  to  the  point,  and  of  such  interest  and  value,  that  we  take 
the  liberty  of  publishing  it  for  the  benefit  of  our  readers." 


3io 


PAR-FOCAL  OCULARS  AND  OBJECTIVES 


[CH.  IX 


TUU  UB* 


"JENA,  June  25th,  1881.  Dear  Sir:  The  question  which  you  ask 
admits  of  a  simple  answer:  In  order  to  change  the  oculars  of  a  micro- 
scope without  changing  the  focus,  of  the  objective,  neither  the  dia- 
phragm nor  the  field-lens  must  come  to  the  same  place  in  the  micro- 
cope  tube,  but  the  anterior  (lower)  focal  points  of  the  ocular  systems 
must  do  this.  In  the  case  of  a  Huygenian  eye-piece,  the  said  anterior 
focus  is  a  virtual  one  situated  above  the  field-lens  at  a  place  Dx,  which 

is  more  distant  from  the  field-lens  than 
the  diaphragm  D.  The  level  of  D*  is 
the  place  where  the  virtual  image  of 
the  diaphragm  appears  to  an  observer 
looking  through  the  field-lens.  Rays 
which  are  required  to  emerge  from  the 
eye-lens  as  parallel  rays  (or  nearly 
parallel)  must  of  course  enter  into  the 
ocular  converging  to  the  point  D*. 
Consequently,  if  different  oculars  are 
inserted  successively  in  such  a  way 
that  the  point  D*  comes  to  the  same 
place  of  the  tube  always,  the  conju- 
gate foci  of  object  and  image  in  the 
objective  remain  unaltered. 

"This  arrangement  and  no  other  one 
fulfills  at  the  same  time  the  other  re- 
quest that  the  amplification  of  the  mi- 
croscope with  different  oculars  should  be  in  exact  inverse  proportion 
of  the  equivalent  focal  length  of  the  oculars. 

"The  position  of  the  point  D*  may  be  easily  calculated  for  every 
ocular.  If  a  is  the  distance  of  the  diaphragm  from  the  field  lens  and 
X  the  focal  length  of  that  lens,  the  distance  of  the  focus  D*  above  the 

diaphram  (i.e.  the  distance  from  D  to  D*)  will  be:  ft  =  — • 

A  —  a 

Hoping  that  these  explanations  will  be  found  satisfactory  for  your 
aim,  I  remain 

Yours  sincerely, 

DR.   E.  ABBE." 


FIG.  184.    PAR-FOCALIZATION  OF 
OCULARS. 

(From  the  Microscopical  Bulletin, 
1886). 


CH.  IX]  PAR-FOCAL  OCULARS  AND  OBJECTIVES  311 

On  page  31  of  the  Bulletin  is  the  following:  "Par-focal  Eye-pieces. 
Referring  to  the  article  in  the  April  issue  of  the  Bulletin,  on  changing 
eye-pieces  without  altering  focus,  etc.,  we  announce  that  we  are  pre- 
pared to  furnish  eye-pieces  as  here  described  with  our  Acme  micro- 
scopes at  a  slight  additional  expense. 

'We  have  named  these  eye-pieces  Par-focal,  meaning  of  equal 
focus,  from  the  Latin  par  (equal)  and  focus" 

For  the  par-focalization  of  objectives,  see  §  74-75. 

COLLATERAL  READING  FOR  CHAPTER  IX 

Optic  Projection,  S.  H.  &  H.  P.  Gage. 

Principles  of  Microscopy,  Sir.  A.  E.  Wright. 

Microscopy,  E.  J.  Spitta. 

The  Microscope  and  its  Revelations,  Carpenter-Dallinger. 

Journal  of  the  Royal  Microscopical  Society. 

Transactions  of  the  American  Microscopical  Society,  especially  the  address  of 
Hon.  J.  D.  Cox,  1884,  pp.  5-39  on  Aperture,  and  1893,  pp.  1-16,  and  A.  C.  Mercer, 
1896,  pp.  321-396. 

C.  W.  Woodworth,  A.  New  Fundamental  Equation  in  Optics.  Science,  N.  S. 
Vol.  XLIII,  pp.  824-825. 

John  C.  Shedd,  The  Index  of  Refraction.  School  Science  and  Mathematics, 
Vol.  VI.,  1906,  pp.  678-680. 

(This  article  gives  a  brief  history  of  the  discovery  of  the  law  of  refraction; 
it  also  discusses  the  ratio  of  velocities  in  different  media,  and  shows  that  the 
coefficient  of  retardation  of  velocity  in  a  transparent  medium  is  the  reciprocal 
of  the  index  of  refraction.) 

According  to  Nelson,  "Par-focal"  oculars  have  been  made  by  Powell  since  1839. 
NELSON,  E.  M.  —  Eye-pieces  for  the  Microscope.  Jour.  Roy.  Micr.  Soc.,  1908, 
p.  149.  See  also  for  other  discussions  of  oculars  by  Nelson,  Same  journal, 
1007,  pp.  525-531;  1900.  PP-  162-169. 


CHAPTER  X 

SLIDES  AND  COVER-GLASSES;  MOUNTING;  ISOLATION;  LABELING 
AND   STORING  MICROSCOPIC  PREPARATIONS;  REAGENTS 

§  485.   Apparatus  and  material  for  Chapter  X.  - 

1.  Glass  slides  for  mounting  micro-          12.    Block  with  holes  for  supporting 
scopic  objects  (fig.  185-187).  vials  (fig.  198). 

2.  Cover-glasses          for         covering          13.   Watch  glasses. 

mounted  objects  (fig.  185-187).  14.   Labels  and  catalogue  card  (§525- 

3.  Glass  dishes  for  storing  slides  and      526). 

covers  (§  492).  15.    Cabinets   and    trays    for   micro- 

4.  Cleaning  mixtures   for  slides  and      scopic  objects  (fig.  204-208). 

covers  (§  488,  492,  497).  16.   Lockers  for  specimens  (fig.  208). 

5.  Micrometer  calipers  and  measurer  17.   Measuring    and   weighing  appa- 
for  slides  and  covers  (fig.  188,  189).  ratus  (fig.  209,  §  536). 

6.  Anatomical  instruments,   forceps,  18.    Bottles  for  containing  the  various 
scissors,  scalpels,  needles.  reagents. 

'7.   Turn-table  for  sealing  cover-glasses  19.    Pipettes  and  simple  microscopes 

and  making  cells  (fig.  191).  (fig.  200-202). 

8.  Centering  card  (fig.  192).  20.   Fixing,   imbedding,   dissociating, 

9.  Moist  chamber  (fig.  199).  mounting  and   staining  agents  (§  534- 

10.  Balsam,  glycerin  jelly  and  shellac      592). 

bottles  (fig.  194-195).  21.     Mounting  media   (balsam,  gly- 

11.  Glass  vials  for  preparations  (fig.      cerin  jelly,  etc.)  (543-547). 
196-197). 

§  486.    Slides,  glass  slides  or  slips,  microscopic  slides  or  slips.  — 

These  are  strips  of  clear  flat  glass  upon  which  microscopic  specimens 
are  usually  mounted  for  preservation  and  ready  examination.  The 
size  that  has  been  almost  universally  adopted  for  ordinary  prepara- 
tions is  25  x  76  millimeters  (i  X  3  inches).  For  rock  sections,  slides 
25  x  45  mm.  or  32  X  32  mm.  are  used;  for  serial  sections,  slides  25 
X  76  mm.,  38  X  76  mm.  or  50  x  76  mm.  are  used.  For  special  pur- 
poses, slides  of  the  necessary  size  are  employed  without  regard  to  any 
conventional  standard. 

Whatever  size  of  slide  is  used,  it  should  be  made  of  clear  glass  and 
the  edges  should  be  ground.  It  is  altogether  false  economy  to  mount 
permanent  microscopic  objects  on  slides  with  unground  edges.  It  is 
unsafe  also,  as  the  unground  edges  are  liable  to  wound  the  hands. 

312 


CH.  X] 


SLIDES    AND    COVER-GLASSES 


313 


Thick  slides  are  preferred  by  many  to  thin  ones.     For  micro- 
chemical  work  Dr.  Chamot  recommends  slides  of  half  the  length  of 


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those  used  in  ordinary  microscopic  work.  From  the  rapidity  with 
which  they  are  destroyed,  he  thinks  the  ground  edges  are  unneces- 
sarily expensive.  He  adds  further:  " It  is  a  great  misfortune  that  the 


314  SLIDES    AND    COVER-GLASSES  [Cn.  X 

colorless  glass  slips  used  in  America  and  so  excellent  for  ordinary 
microscopic  work  should  be  easily  attacked  by  all  liquids;  even  water 
extracts  a  relatively  enormous  amount  of  alkalies  and  alkaline  earths. 
The  slips  of  greenish  glass,  while  not  as  neat  or  desirable  for  general 
microscopy,  seem  to  be  decidedly  more  resistant,  and  are  therefore 
preferable."  Transparent  celluloid  slides  are  recommended  by 
Behrens  for  work  where  hydrofluoric  acid  and  its  derivatives  are  to 
be  examined.  (Chamot,  Jour.  Appl.  Micr.,  vol.  iii,  p.  793.  Chemical 
Microscopy,  p.  123-124). 

§  487.  Thickness  of  slides  for  special  purposes.  —  It  is  very  impor- 
tant to  observe  strictly  the  requirements  for  the  thickness  of  slide  for 
special  purposes.  As  pointed  out  in  discussing  the  dark-ground  con- 
denser (§  125-127),  the  slide  must  be  thin  enough  so  that  the  focus 
of  the  condenser  will  be  just  above  the  upper  surface  where  the  object 
is  mounted.  If  the  slide  is  too  thick  the  focus  will  be  beneath  the 
object  and  the  best  light  cannot  be  obtained.  So  likewise  with  the 
best  achromatic  condensers,  especially  when  used  as  homogeneous 
immersion  condensers  (§  471),  if  the  slide  is  too  thick  the  focus  of  the 
condenser  will  fall  below  the  object  and  the  best  and  most  critical 
images  cannot  be  obtained. 

It  is  better  to  use  a  slide  thinner  than  the  maximum  permissible 
and  plenty  of  homogeneous  liquid  between  the  slide  and  the  condenser, 
then  the  condenser  can  be  lowered  until  its  focus  is  upon  the  object. 
This  applies  equally  with  the  dark-ground  condenser.  For  getting  the 
thickness  of  the  slides,  use  the  micrometer  calipers  or  the  cover-glass 
measurer  (fig.  188-189). 

§  488.  Cleaning  slides  for  ordinary  use.  —  Place  new  slides  that 
are  to  be  wiped  at  once  in  a  glass  vessel  of  distilled  water  contain- 
ing 5%  ammonia.  For  wiping  the  slides  use  a  lintless  towel  or  a 
well-washed  linen  towel.  One  may  avoid  large  wash  bills  by  using 
absorbent  gauze  (§  488a). 

In  handling  the  slides  grasp  them  by  the  edges.  Cover  the  fingers 
of  the  right  hand  with  the  wiping  towel  or  the  gauze  and  rub  both 
faces  with  it.  When  wiped  thoroughly  dry,  place  the  slide  in  a  dry 
glass  jar  or  for  larger  numbers  use  a  museum  jar  (fig.  214).  Soap  and 
water  are  also  recommended  for  new  slides. 


CH.  X]  CLEANING    SLIDES    AND    COVER-GLASSES  315 

Alcohol  of  50%  to  82%  is  also  excellent  for  cleaning  new  slides, 
and  for  slides  which  have  been  freed  from  mounting  media  by  boil- 
ing (§  489)  after  a  thorough  rinsing  in  clean  water. 

§  488a.  Absorbent  gauze  and  lintless  towels.  —  The  gauze  mentioned  is 
No.  10,  "  Sterilized  absorbent  gauze,"  of  the  Griswoldville  Mfg.  Co.  of  New  York. 
It  is  sometimes  cr  ll?d  bleached  cheese  cloth.  In  the  author's  laboratory  it  is  cut 
into  pieces,  I,  I,  ^  of  a  yard.  When  a  piece  is  soiled  it  is  thrown  away.  There 
has  recently  appeared  specially  prepared  towels  for  wiping  glass  etc.,  which  are 
called  "  lintless,"  as  practically  no  lint  is  left  on  the  wiped  object.  These  are  fur- 
nished by  Johnson  &  Johnson  of  New  York,  and  cost  about  15  cents  each  in  a  size 
42  x  90  cm. 

§  489.  Cleaning  used  slides.  —  If  only  watery  substances  or  gly- 
cerin or  glycerin  jelly  have  been  used  one  may  soak  the  slides  over- 
night in  ammonia  water,  then  change  the  water  for  fresh  and  wipe 
as  described  in  §  488. 

When  balsam  or  other  resinous  media  (§  543)  have  been  used  it  is 
best  to  heat  the  slides  over  a  Bunsen  flame  and  remove  the  cover- 
glass.  Place  the  covers  in  cleaning  mixture  (§  497) .  The  slide  may 
also  be  placed  in  cleaning  mixture  or  in  some  hot  water  containing 
10%  gold  dust  or  other  strong  alkaline  cleaner.  When  the  metal 
basin  —  preferably  an  agateware  basin  —  is  two-thirds  full  of  the 
slides,  heat  until  the  water  comes  to  a  boil.  Then  let  it  cool.  Add 
fresh  water  and  most  of  the  slides  may  be  wiped  clean. 

If  dichromate  cleaning  mixture  is  used  the  best  method  is  to  have 
a  museum  jar  of  it  and  drop  the  slides  in  as  they  are  rejected,  or  a 
large  number  at  once,  as  is  most  convenient.  It  may  require  a  week 
or  more  to  clean  the  slides  with  cleaning  mixture.  As  this  is  a  very 
corrosive  mixture  for  metals  use  only  glass  dishes  in  dipping  into 
it.  When  the  slides  are  freed  from  balsam,  etc.,  pour  off  the  clean- 
ing mixture  into  another  glass  vessel  and  allow  a  stream  of  water  to 
flow  over  the  slides  until  all  the  cleaning  mixture  has  been  washed 
away.  Then  add  distilled  water  and  wipe  the  slides  from  that.  Any 
slides  still  not  freed  from  the  balsam  should  be  put  back  into  the 
cleaning  mixture.  Apparently  the  slides  are  not  injured  by  a  pro- 
longed stay  in  the  mixture. 

§  490.  Cleaning  slides  for  special  uses.  —  In  making  blood  films, 
for  micro-chemistry  and  whenever  an  even  film  is  desired  every  particle 


3i6  CLEANING    SLIDES    AND    COVER-GLASSES  [Cn.  X 

of  oily  substance  must  be  removed.  The  slides  should  be  placed  in 
the  dichromate  cleaning  mixture  (§  497)  one  day  or  more,  thoroughly 
washed  with  clean  water  and  then  in  distilled  water,  or  in  50%  to 
75  %  alcohol.  They  are  taken  from  the  water  or  alcohol  and  wiped 
dry  as  needed.  In  wiping  keep  two  or  more  layers  of  the  absorbent 
gauze  over  the  fingers.  Only  one  slide  is  wiped  with  each  piece  of 
gauze.  The  surface  to  touch  the  slides  should  never  have  been 
touched  by  the  hands,  for  a  minute  amount  of  oily  substance  leaves  a 
stratum  on  the  slide  which  causes  the  liquids  used  to  heap  up  instead  of 
flowing  out  perfectly  flat.  That  is,  the  slide  is  wet  with  difficulty 
and  the  liquid  instead  of  forming  a  film  tends  to  assume  the  spheroidal 
state.  Sometimes  new  gauze  or  other  j:loth  used  may  not  be  wholly 
free  from  oily  substance,  or  the  soap  was  not  wholly  eliminated  in 
washing.  Such  wiping  cloths  will  not  make  the  slides  ready  for  good 
films.  Some  workers  soak  the  gauze  in  sulphuric  ether  to  remove  the 
last  traces  of  oily  substance.  This  is  done  more  especially  in  cleaning 
cover-glasses  for  films  (see  below).  Burnett,  p.  22,  in  speaking  of  blood 
smears,  says:  "The  slides  should  be  thoroughly  clean.  Unused  slides 
may  be  cleaned  in  strong  soap  or  'gold  dust'  solution  well  rinsed 
in  water,  then  placed  in  alcohol  from  which  they  are  wiped  and 
polished." 

As  intimated  above,  the  best  way  to  tell  when  slides  or  covers  are 
free  from  a  surface  film  is  to  drop  some  water  on  the  surface  and  then 
hold  the  slide  or  cover  nearly  vertical.  If  the  surface  is  clean  the 
water  will  run  over  the  slide,  leaving  a  smooth  wet  track.  If  a  film 
of  oily  substance  is  present  the  water  will  crawl  and  form  ridges  or 
droplets  and  not  leave  a  smooth  wet  surface.  Sometimes  it  is  almost 
impossible  to  get  a  slide  so  that  a  smooth,  even  film  of  blood  or  other 
liquid  can  be  made  upon  it.  According  to  Chamot  (p.  124),  such  slides 
may  be  rendered  suitable  for  use,  in  many  cases,  by  passing  them 
slowly  through  the  Bunsen  flame.  Cover-glasses  are  also  rendered 
usable  by  the  same  method  when  they  are  refractory  after  wiping 
carefully. 

§  491.  Cover-glasses  or  covering  glasses.  —  These  are  circular 
or  quadrangular  pieces  of  thin  glass  used  for  covering  and  protecting 
microscopic  objects.  They  should  be  very  thin,  o.io  to  0.25  milli- 


CH.  X]  CLEANING    SLIDES    AND    COVER-GLASSES  317 

meter.  It  is  better  never  to  use  a  cover-glass  over  o.  20  mm.  thick,  then 
the  preparation  may  be  studied  with  a  2  mm.  oil  immersion  as  well  as 
with  lower  objectives.  Except  for  objects  wholly  unsuited  for  high 
powers,  it  is  a  great  mistake  to  use  cover-glasses  thicker  than  the 
working  distance  of  a  homogeneous  objective  (§  76).  Indeed,  if  one 
wishes  to  employ  high  powers,  the  thicker  the  section  the  thinner 
should  be  the  cover-glass. 

The  cover-glass  should  always  be  considerably  larger  than  the  object 
over  which  it  is  placed. 

§  492.  Cleaning  cover-glasses  for  ordinary  use.  —  Covers  may 
be  cleaned  well  by  placing  them  in  82  %  or  95  %  alcohol  containing 
hydrochloric  acid  one  per  cent.  They  may  be  wiped  almost  imme- 
diately. 

Remove  a  cover  from  the  alcohol,  grasping  by  the  edge  with  the  left 
thumb  and  index.  Cover  the  right  thumb  and  index  with  some  clean 
gauze  or  other  absorbent  cloth;  grasp  the  cover  between  the  thumb 
and  index  and  rub  the  surfaces,  keeping  the  thumb  and  index  well 
opposed  on  directly  opposite  faces  of  the  cover  so  that  no  strain  will 
come  on  it,  otherwise  the  cover  is  liable  to  be  broken. 

When  a  cover  is  dry  hold  it  up  and  look  through  it  toward  some 
dark  object.  The  cover  will  be  seen  partly  by  transmitted  and 
partly  by  reflected  light,  and  any  cloudiness  will  be  easily  detected. 
If  the  cover  does  not  look  clear,  breathe  on  the  faces  and  wipe  again. 
If  it  is  not  possible  to  get  a  cover  clean  in  this  way  it  should  be  put 
again  into  the  cleaning  mixture. 

As  the  covers  are  wiped  put  them  in  a  clean  shell  vial  (fig.  196), 
glass  box  or  Petri  dish.  Handle  them  always  by  their  edges,  or  use 
fine  forceps.  Do  not  put  the  fingers  on  the  faces  of  the  covers,  for 
that  will  surely  cloud  them. 

§  493.  Cleaning  cover-glasses  for  special  uses.  —  As  with  slides, 
covers  intended  for  films  or  other  purposes  where  the  least  particles  of 
oily  substance  must  be  removed,  are  best  put  one  by  one  into  dichro- 
mate  cleaning  mixture  (§  497).  After  a  day  or  more  this  is  poured 
off  and  a  stream  of  fresh  water  allowed  to  run  on  the  covers  until  all 
the  cleaning  mixture  is  removed.  Then  distilled  water  is  added  and 
allowed  to  stand  a  few  minutes.  This  is  poured  off  and  82  %  or  95  % 


CLEANING    SLIDES    AND    COVER-GLASSES  [Cn.  X 


32nds. 
1    .0312 
3   .0937 
5  .1562 
7 .2187 
9 .2812 
11  .3437 
13.4062 
15.4687 


1-8  .125 
1-4.  .2  5  0 
3-8  .375 
ICths. 
1    .0625 
3    .1875 
5    .3125 
7  .4375 


FIG.  1 88.     MICROMETER  CALIPERS  FOR  MEASURING 
THE  THICKNESS  OF  SLIDES  AND  COVER-GLASSES. 

(From  the  Catalogue  of  the  Brown  &  Sharpe  Manu- 
facturing Company). 


alcohol  added.  The 
covers  remain  in  this 
until  needed.  In  wip- 
ing use  the  precau- 
tions given  with  slides 

(§49°)- 

Test  for  the  proper 
cleanliness  as  for  slides 
(§  490),  and  remember 
the  advantage  of  heat- 
ing the  cover-glass  in 
a  Bunsen   or   alcohol 
flame  to  render  it  cap- 
able of  receiving  a  smooth  and  even  film  of  blood  or  other  aqueous 
liquid. 

§  494.  Cleaning  large  cover-glasses.  —  For  serial  sections  and 
especially  large  sections,  large 
quadrangular  covers  are  used. 
These  are  to  be  put  one  by  one 
into  a  cleaning  mixture  as  for 
the  smaller  covers  and  treated 
in  every  way  the  same.  In  wip- 
ing them  one  may  proceed  as  for 
the  small  covers,  but  special 
care  is  necessary  to  avoid  break- 
ing them.  It  is  desirable  that 
these  large  covers  should  be 
thin  —  not  over  0.15-0.20  mm., 
otherwise  high  objectives  can- 


FIG.  189.    MEASURER  FOR  SLIDES  AND 
COVER-GLASSES. 


not   be   used    in    studying   the 
preparations. 

§  495.  Measuring  the  thick- 
ness of  cover-glasses.  —  It  is  of 
great  advantage  to  know  the  ex- 
act thickness  of  the  cover-glass 
on  an  object;  for,  (a)  in  study- 


from  the  figure  in  Zeiss 
Catalogue). 

Cover  A  cover-glass  between  the  jaws  (f)  • 

J  Jaws  between  which  the  object  to 
be  measured  is  placed. 

H  Hand  which  points  to  the  gradua- 
tions on  the  face  indicating  the  fractions 
of  a  millimeter  or  inch  that  represents  the 
thickness  of  the  object  between  the  jaws. 

L  Lever  by  which  the  jaws  are  opened 
to  receive  the  object. 


CH.  X]  MEASURING    SLIDES    AND    COVERS  319 

ing  the  preparation  one  would  not  try  to  use  objectives  of  a  shorter 
working  distance  than  the  thickness  of  the  cover  (§  76) ;  (b)  hi  using 
adjustable  objectives  with  the  collar  graduated  for  different  thick- 
nesses of  cover,  the  collar  can  be  set  at  a  favorable  point  without  loss 
of  time;  (c)  for  unadjustable  objectives  the  thickness  of  cover  may 
be  selected  corresponding  to  that  for  which  the  objective  was  cor- 
rected (§  460) .  Furthermore,  if  there  is  a  variation  from  the  stand- 
ard, one  may  remedy  it,  in  part  at  least,  by  lengthening  the  tube  if  the 
cover  is  thinner,  and  shortening  it  if  the  cover  is  thicker  than  the 
standard  (§462). 

Among  the  so-called  No.  i  cover-glasses  of  the  dealers  in  micro- 
scopical supplies,  the  writer  has  found  covers  varying  from  o.io  mm. 
to  0.35  mm.  To  use  cover-glasses  of  so  wide  a  variation  in  thickness 
without  knowing  whether  one  has  a  thick  or  thin  one  is  simply  to 
ignore  the  fundamental  principles  by  which  correct  microscopic 
images  are  obtained. 

From  information  supplied  by  Mr.  Edward  Pennock  the  thickness 
of  various  cover-glasses  should  be  within  the  following  limits: 

No.  i  cover-glasses.  .  .  .0.12  to  0.18  mm. 

No.  2 0.18  to  0.25  mm. 

No.  3 0.25  to  0.50  mm. 

No.  o o.io  mm.  slightly  more  or  less. 

In  general  cover-glasses  thinner  than  the  minimum  (0.12  mm.)  of 
No.  i,  actual  measurement,  will,  as  stated  above,  usually  show 
a  much  wider  variation. 

It  is  then  strongly  recommended  that  every  preparation  shall  be 
covered  with  a  cover-glass  whose  thickness  is  known,  and  that  this 
thickness  be  indicated  in  some  way  on  the  preparation. 

§  496.  Cover-glass  measures,  testers,  or  gauges.  —  For  the  pur- 
pose of  measuring  cover-glasses  there  are  two  very  excellent  pieces  of 
apparatus.  The  micrometer  calipers  (fig.  188),  used  chiefly  in  the 
mechanic  arts,  are  convenient  and  from  their  size  are  easily  carried  in 
the  pocket.  The  cover-glass  measurer  specially  designed  for  the 
purpose  is  shown  in  fig.  189,  by  which  covers  may  be  more  rapidly 
measured  than  with  the  calipers. 


320  MEASURING    SLIDES    AND    COVERS  [Cn.  X 

With  these  measures  or  gauges  one  should  be  certain  that  the  index 
stands  at  zero  when  at  rest.  If  the  index  does  not  stand  at  zero  it 
should  be  adjusted  at  that  point,  otherwise  the  readings  will  not  be 
correct. 

As  the  covers  are  measured,  the  different  thicknesses  should  be  put 
into  different  glass  boxes  and  properly  labeled.  Unless  one  is  striving 
for  the  most  accurate  possible  results,  cover-glasses  not  varying 
more  than  0.06  mm.  may  be  put  in  the  same  box.  For  example,  if 
one  takes  0.15  mm.  as  a  standard,  covers  varying  0.03  mm.  on  each 
side  may  be  put  into  the  same  box.  In  this  case  the  box  would  con- 
tain covers  of  0.12,  0.13,  0.14,  0.15,  0.16,  0.17,  and  0.18  mm. 

§  497.  Bichromate  cleaning  mixture  for  glass.  —  The  cleaning 
mixture  used  for  cleaning  slides  and  cover-glasses  is  that  commonly 
used  in  chemical  laboratories:  (Dr.  G.  C.  Caldwell's  Laboratory 
Guide  in  Chemistry). 

Bichromate  of  potash  (K^CraO?) 200  grams 

Water,  distilled  or  ordinary 800  cc. 

Sulphuric  acid  (H^SCX) 1 200  cc. 

As  great  heat  is  developed  in  the  reaction  on  mixing  the  sulphuric 
acid  with  the  watery  solution  of  dichromate,  it  is  necessary  to  use  heat- 
resisting  vessels.  The  best  so  far  employed  are  those  made  of  pyrex 
glass.  Use  ordinary  tap  water  and  the  commercial  dichromate  and 
strong  sulphuric  acid.  Chemically  pure  ingredients  are  not  demanded. 

Dissolve  the  dichromate  in  the  water  by  the  aid  of  heat.  Use  for 
this  an  agate  dish.  Now  place  the  pyrex  dish  in  the  sink  on  some 
asbestos  or  a  piece  of  board.  Pour  the  warm  solution  of  dichromate 
into  the  pyrex  dish,  and  then  add  the  sulphuric  acid,  stirring  the 
liquid  with  a  glass  rod.  The  reaction  is  so  great  that  the  liquid  will 
boil  violently.  An  abundance  of  chromic  acid  crystals  will  form  as 
the  sulphuric  acid  is  added.  Let  the  pyrex  dish  remain  in  the  sink 
until  the  cleaning  mixture  is  cool  and  then  pour  it  into  a  glass- 
stoppered  bottle  for  storage. 

If  the  dichromate  is  well  pulverized  it  can  be  put  directly  into  the 
pyrex  dish  with  the  requisite  amount  of  water,  and  the  sulphuric 
acid  added  as  directed. 


CH.  X]  MOUNTING    MICROSCOPIC    OBJECTS  321 

This  is  an  excellent  cleaning  mixture  and  is  practically  odorless. 
It  is  exceedingly  corrosive  and  must  be  kept  in  glass  vessels.  It 
may  be  used  more  than  once,  but  when  the  color  changes  markedly 
from  that  seen  in  the  fresh  mixture  it  should  be  thrown  away.  An 
indefinite  sojourn  of  the  slides  and  covers  in  the  cleaner  does  not  seem 
to  injure  them. 

MOUNTING,  AND  PERMANENT  PREPARATION  OF  MICROSCOPIC 

OBJECTS 

§  498.  Mounting  a  microscopic  object  is  so  arranging  it  upon 
some  suitable  support  (glass  slide)  and  in  some  suitable  mounting 
medium  that  it  may  be  satisfactorily  studied  with  the  microscope. 

The  cover-glass  on  a  permanent  preparation  should  always  be  consid- 
erably larger  than  the  object;  and  where  several  objects  are  put  under 
one  cover-glass,  as  with  serial  sections,  it  is  false  economy  to  crowd  them 
too  closely  together. 

§  499.  Temporary  mounting ;  normal  fluids.  —  In  a  great  many 
cases  objects  do  not  need  to  be  preserved;  they  are  then  mounted 
in  any  way  to  enable  one  best  to  study  them,  and  after  the  study  the 
cover-glass  is  removed,  and  the  slide  cleaned  for  future  use.  In  the 
study  of  living  objects,  of  course  only  temporary  preparations  are 
possible.  With  amoebae,  white  blood  corpuscles,  and  many  other 
objects,  both  animal  and  vegetable,  the  living  phenomena  can  best  be 
studied  by  mounting  them  in  the  natural  medium.  That  is,  for  amce- 
bse,  in  the  water  in  which  they  are  found;  for  the  white  blood  cor- 
puscles, a  drop  of  blood  is  used  and,  as  the  blood  soon  coagulates,  they 
are  in  the  serum.  Sometimes  it  is  not  easy  or  convenient  to  get  the 
natural  medium;  then  some  liquid  that  has  been  found  to  serve  in 
place  of  the  natural  medium  is  used.  For  many  things,  water  with 
a  little  common  salt  (water  1000  cc.,  common  salt  7.5  grams)  is 
employed.  This  is  the  so-called  isotonic  or  normal  salt  or  saline 
solution.  For  the  ciliated  cells  from  frogs  and  other  amphibia, 
nothing  has  been  found  so  good  as  human  spittle.  Whatever  is  used, 
the  object  is  put  on  the  middle  of  the  slide  and  a  drop  of  the  mounting 
medium  added,  and  then  the  cover-glass.  The  cover  is  best  put  on 
with  fine  forceps,  as  shown  in  fig.  190.  After  the  cover  is  in  place,  if 


322  MOUNTING    MICROSCOPIC    OBJECTS  [Cn.  X 

the  preparation  is  to  be  studied  for  some  time,  it  is  better  to  avoid 
currents  and  evaporation  by  painting  a  ring  of  castor  oil  around  the 
cover  in  such  a  way  that  part  of  the  ring  will  be  on  the  slide  and  part 
on  the  cover  (fig.  204). 

It  cannot  be  too  strongly  emphasized  that  if  one  is  to  study  living 
or  fresh  tissues  they  must  be  mounted  in  a  liquid  which  will  not  injure 
them.  The  liquid  in  which  they  are  naturally  found  is  of  course  the 
most  nearly  normal  of  any,  and  should  be  always  used  when  possible. 
Water  seems  a  very  bland  and  harmless  liquid,  but  it  has  a  very  de- 
cidedly injurious  effect  on  living  tissues  which  are  normally  bathed  by 

the  fluids  of  the  body,  for 
forctji/       they  always  contain  salts 
and  colloid  material.  Dis- 
tilled water  is  more  de- 


FIG.  190.    FINE  FORCEPS  FOR  HANDLING  COVER-    leterious  than  tap  water 
GLASSES  AND  OTHER  DELICATE  OBJECTS.  because    k    contains    no 

salts.  It  would  be  deleterious  to  water  organisms,  because  all  natural 
waters  contain  a  greater  or  less  quantity  of  organic  and  inorganic 
substances  in  solution.  In  examining  water  organisms  use  the  water 
in  which  they  are  found.  If  the  water  supply  of  a  city  or  town  has 
a  filtration  plant  the  water  is  likely  to  be  unsuitable  for  raising  water 
forms  like  salamander  embryos,  and  the  embryos  of  the  frogs  and 
toads,  besides  many  other  water  forms.  One  must  take  the  trouble  to 
get  the  water  from  the  natural  breeding  places  if  the  embryos  are  to 
be  successfully  raised  in  a  laboratory.  (See  also  §  520-521,  584.) 

§  500.  Permanent  mounting.  —  There  are  three  great  methods  of 
making  permanent  microscopic  preparations.  Special  methods  of 
procedure  are  necessary  to  mount  objects  successfully  in  each  of  these 
ways.  The  best  mounting  medium  and  the  best  method  of  mounting 
in  a  given  case  can  only  be  determined  by  experiment.  In  most 
cases  some  previous  observer  has  already  made  the  necessary  experi- 
ments and  furnished  the  desired  information. 

The  three  methods  are  the  following: 

(1)  Dry  or  in  air  (§  501-504). 

(2)  In  some  medium  miscible  with  water,  as  glycerin  or  glycerin 
,    i  jelly  (§  505-509)- 


CH.  X]  MOUNTING    MICROSCOPIC    OBJECTS  323 

(3)   In  some  resinous  medium  like   Canada  balsam,  damar,  etc 


§  501.  Mounting  dry  or  in  air.  —  The  object  should  be  thoroughly 
dry.  If  any  moisture  remains  it  is  liable  to  cloud  the  cover-glass,  and 
the  specimen  may  deteriorate.  As  the  specimen  must  be  sealed,  it 
is  necessary  to  prepare  a  cell  slightly  deeper  than  the  object  is  thick. 
This  is  to  support  the  cover-glass,  and  also  to  prevent  the  running  in 
by  capillarity  of  the  sealing  mixture. 

Order  of  procedure  in  mounting  objects  dry  or  in  air. 

1.  A  cell  of  some  kind  is  prepared.     It  should  be  slightly  deeper 
than  the  object  is  thick  (§  503). 

2.  The  object  is  thoroughly  dried  (desiccated)  either  in  dry  air  or  by 
the  aid  of  gentle  heat. 

3.  If  practicable  the  object  is  mounted  on  the  cover-glass;  if  not  it 
is  placed  in  the  bottom  of  the  cell. 

4.  The  slide  is  warmed  till  the  cement  forming  the  cell  wall  is 
somewhat  sticky,  or  a  very  thin  coat  of  fresh  cement  is  added;    the 
cover  is  warmed  and  put  on  the  cell  and  pressed  down  all  around  till 
a  shining  ring  indicates  its  adherence. 

5.  The  cover-glass  is  sealed. 

6.  The  slide  is  labeled. 

7.  The  preparation  is  catalogued  and  safely  stored. 

§  502.  Example  of  mounting  dry,  or  in  air.  —  Prepare  a  shallow 
cell  and  dry  it  (§  503).  Select  a  clean  cover-glass  slightly  larger  than 
the  cell.  Pour  upon  the  cover  a  drop  of  10%  solution  of  salicylic 
acid  in  95  %  alcohol.  Let  it  dry  spontaneously.  Warm  the  slide  till 
the  cement  ring  or  cell  is  somewhat  sticky;  then  warm  the  cover 
gently  and  put  it  on  the  cell,  crystals  down.  Press  on  the  cover  all 
around  the  edge,  seal,  label,  and  catalogue. 

A  preparation  of  mammalian  red  blood  corpuscles  may  be  satis- 
factorily made  by  spreading  a  very  thin  layer  of  fresh  blood  on  a 
cover  with  the  end  of  a  slide.  After  it  is  dry,  warm  gently  to  remove 
the  last  traces  of  moisture  and  mount  blood  side  down,  precisely  as  for 
the  crystals.  One  can  get  the  blood  as  directed  for  the  Micro-spectro- 
scopic  work  (§413). 


324  MOUNTING    MICROSCOPIC    OBJECTS  [Cn.  X 

§  503.  Preparation  of  mounting  cells.  —  (A)  Thin  cells.  These 
are  most  conveniently  made  of  some  of  the  cements  used  in  micros- 
copy. Shellac  is  one  of  the  best  and  most  generally  applicable.  To 
prepare  a  shellac  cell  place  the  slide  on  a  turn-table  (fig.  191)  and 
center  it,  that  is,  get  the  center  of  the  slide  over  the  center  of  the  turn- 
table. Select  a  guide  ring  on  the  turn-table  which  is  a  little  smaller 
than  the  cover-glass  to  be  used,  take  the  brush  from  the  shellac,  being 
sure  that  there  is  not  enough  cement  adhering  to  it  to  drop.  Whirl 


FIG.  191.    TURN-TABLE  FOR  MAKING  CELLS  AND  FOR  SEALING  COVER-GLASSES. 

Hand  Rest  The  metal  plate  supporting  the  hand  that  holds  the  brush.  It  can 
be  raised  or  lowered  by  means  of  the  screw  underneath  (s). 

sc     Spring  clips  for  holding  the  slide  in  place. 

gc     Guide  circles  to  aid  in  centering  the  slide  or  the  mounted  object. 

me  Milled  circular  disc  by  which  the  turn-table  is  whirled  when  the  ring  of 
cement  is  being  painted  around  the  cover-glass  or  the  mounting  cell. 

the  turn- table  and  hold  the  brush  lightly  on  the  slide  just  over  the 
guide  ring  selected.  An  even  ring  of  cement  should  result.  If  it  is 
uneven,  the  cement  is  too  thick  or  too  thin,  or  too  much  was  on  the 
brush.  After  a  ring  is  thus  prepared  remove  the  slide  and  allow  the 
cement  to  dry  spontaneously,  or  heat  the  slide  in  some  way.  Before 
the  slide  is  used  for  mounting,  the  cement  should  be  so  dry  when 
it  is  cold  that  it  does  not  dent  when  the  finger  nail  is  applied  to  it. 

A  cell  of  considerable  depth  may  be  made  with  the  shellac  by  adding 
successive  layers  as  the  previous  one  dries. 

(B)  Deep  cells  are  sometimes  made  by  building  up  cement  cells,  but 
more  frequently,  paper,  wax,  glass,  hard  rubber,  or  some  metal  is 
used  for  the  main  part  of  the  cell.  Paper  rings,  block  tin  or  lead 


CH.  X]  MOUNTING    MICROSCOPIC    OBJECTS  325 

rings  are  easily  cut  out  with  gun  punches.     These  rings  are  fastened  to 
the  slide  by  using  some  cement  like  the  shellac. 

§  504.  Sealing  the  cover-glass  for  dry  objects  mounted  in  cells.  - 
When  an  object  is  mounted  in  a  cell,  the  slide  is  warmed  until  the 
cement  is  slightly  sticky  or  a  very  thin  coat  of  fresh  cement  is  put  on. 
The  cover-glass  is  warmed  slightly  also,  both  to  make  it  stick  to  the 
cell  more  easily,  and  to  expel  any  remaining  moisture  from  the 
object.  When  the  cover  is  put  on,  it  is  pressed  down  all  around  over 
the  cell  until  a  shining  ring  appears,  showing  that  there  is  an  intimate 
contact.  In  doing  this  use  the  convex  part  of  the  fine  forceps  or  some 
other  blunt,  smooth  object;  it  is  also  necessary  to  avoid  pressing  on  the 
cover  except  immediately  over  the  wall  of  the  cell  for  fear  of  break- 
ing the  cover.  When  the  cover  is  in  contact  with  the  wall  of  cement 
all  around,  the  slide  should  be  placed  on  the  turn-table  and  carefully 
arranged  so  that  the  cover-glass  and  cell  wall  will  be  concentric  with 
the  guide  rings  of  the  turn-table.  Then  the  turn-table  is  whirled  and 
a  ring  of  fresh  cement  is  painted,  half  on  the  cover  and  half  on  the  cell 
wall  (fig.  204) .  If  the  cover-glass  is  not  in  contact  with  the  cell  wall 
at  any  point  and  the  cell  is  shallow,  there  will  be  great  danger  of  the 
fresh  cement  running  into  the  cell  and  injuring  or  spoiling  the  prepara- 
tion. When  the  cover-glass  is  properly  sealed,  the  preparation  is  put 
in  a  safe  place  for  the  drying  of  the  cement.  It  is  advisable  to  add 
a  fresh  coat  of  cement  occasionally. 

§  505.  Mounting  objects  in  media  miscible  with  water.  —  Many 
objects  are  so  greatly  modified  by  drying  that  they  must  be  mounted 
in  some  medium  other  than  air.  In  some  cases  water  with  something 
in  solution  is  used.  Glycerin  of  various  strengths  and  glycerin 
jelly  are  also  much  employed.  All  these  media  keep  the  object  moist 
and  therefore  in  a  condition  resembling  the  natural  one.  The  object 
is  usually  and  properly  treated  with  gradually  increasing  strengths  of 
glycerin  or  fixed  by  some  fixing  agent  before  being  permanently 
mounted  in  strong  glycerin  or  either  of  the  other  media. 

In  all  of  these  different  methods,  unless  glycerin  of  increasing 
strengths  has  been  used  to  prepare  the  tissue,  the  fixing  agent  is  washed 
away  with  water  before  the  object  is  finally  and  permanently  mounted 
in  either  of  the  media. 


326 


MOUNTING    MICROSCOPIC    OBJECTS 


[CH.  X 


For  glycerin  jelly  no  cell  is  necessary  unless  the  object  has  a  con- 
siderable  thickness. 

§  506.    Order   of   procedure   in   mounting    objects  in   glycerin  — 

1.  A  cell  must  be  prepared  on  the  slide  if  the  object  is  of  considerable 
thickness  (§  503). 

2.  A  suitably  prepared  object  is  placed  on  the  center  of  a  clean 
slide,  and  if  no  cell  is  required  a  centering  card  is  used  to  facilitate 
the  centering  (fig.   192). 


11 

Wim 

'.'//;//,  I'L 


m 


FIG.  192.    GUIDE  CARD  TO  AID  IN  MOUNTING  OBJECTS  NEATLY. 

3.  A  drop  of  pure  glycerin  is  poured  upon  the  object,  or  if  a  cell  is 
used,  enough  to  fill  the  cell  and  a  little  more. 

4.  In  putting  on  the  cover-glass  it  is  grasped  with  fine  forceps  and 
the  underside  breathed  on  to  slightly  moisten  it  so  that  the  glycerin 
will  adhere ;   then  one  edge  of  the  cover  is  put  on  the  cell  or  slide  and 
the  cover  gradually  lowered  upon  the  object.     The  cover  is  then  gently 
pressed  down.     If  a  cell  is  used,  a  fresh  coat  of  cement  is  added  before 
mounting. 

5.  The  cover-glass  is  sealed. 

6.  The  slide  is  labeled. 

7.  The  preparation  is  catalogued  and  safely  stored. 

§  507.   Order  of  procedure  in  mounting  objects  in  glycerin  jelly, 
i.   Unless  the  object  is  quite  thick  no  cell  is  necessary  with  glycerin 
jelly. 


CH.  X] 


MOUNTING    MICROSCOPIC    OBJECTS 


327 


2.  A  slide  is  gently  warmed  and  placed  on   the  centering  card 
(fig.  192)  and  a  drop  of  warmed  glycerin  jelly  is  put  on  its  center.    The 
suitably  prepared  object  is  then  arranged  in  the  center  of  the  slide. 

3.  A  drop  of  the  warm  glycerin  jelly  is  then 
put  on  the  object,  or  if  a  cell  is  used  it  is  filled 
with  the  medium. 

4.  The  cover-glass  is  grasped  with  fine  forceps, 
the  lower  side  breathed  on  and  then  gradually 
lowered  upon  the  object  and  gently  pressed  down. 

5.  After  mounting,  the  preparation  is  left  flat 
in  some  cool  place  till  the  glycerin  jelly  sets;  then 
the  superfluous   amount  is  scraped   and  wiped 
away   and   the    cover-glass   sealed  with   shellac 
(§  508). 

6.  The  slide  is  labeled. 

7.  The  preparation  is  catalogued  and  safely 
stored. 

§  508.  Sealing  the  cover-glass  when  no  cell  is 
used.  —  (A)  For  glycerin-mounted  specimens.  The 
superfluous  glycerin  is  wiped  away  as  carefully  as 
possible  with  a  moist  cloth;  then  four  minute 
drops  of  cement  are  placed  at  the  edge  of  the 
cover  (fig.  193)  and  allowed  to  harden  for  half  an 
hour  or  more.  These  will  anchor  the  cover-glass ; 
then  the  preparation  may  be  put  on  the  turn- 
table and  ringed  with  cement  while  whirling  the 
turn-table. 

(B)  For  objects  in  glycerin  jelly,  Farrants' 
solution  or  a  resinous  medium.  The  mounting 
medium  is  first  allowed  to  harden;  then  the 
superfluous  medium  is  scraped  away  as  much  as 
possible  with  a  knife,  and  then  removed  with  a 
cloth  moistened  with  water  for  the  glycerin  jelly  and  Farrants'  solu- 
tion or  with  alcohol,  chloroform  or  turpentine,  etc.,  if  a  resinous  me- 
dium is  used.  Then  the  slide  is  put  on  a  turn-table  and  a  ring  of  the 
shellac  cement  added. 


FIG.  193.  ANCHOR- 
ING THE  COVER- 
GLASS  AND  IRRIGA- 
TION. 

A  This  is  to  show 
the  method  of  an- 
choring a  cover-glass 
by  means  of  four 
drops  of  cement  so 
that  it  may  be  sealed 
on  the  turn-table 
without  danger  of 
displacing  the  cover. 

B  Method  of  ir- 
rigation. A  drop  of 
the  irrigating  liquid 
is  put  on  one  side  of 
the  cover-glass  and 
a  piece  of  absorbent 
paper  on  the  other. 
As  shown  by  the 
arrow  the  liquid  is 
drawn  through.  As 
it  spreads  more  or 
less  on  both  sides  all 
degrees  of  the  reac- 
tion produced  by  the 
liquid  can  be  found 
in  the  same  prepara- 
tion. 


328 


MOUNTING    MICROSCOPIC    OBJECTS 


[CH.  X 


(C)  Balsam  preparations  may  be  sealed  with  shellac  as  soon  as  they 
are  prepared,  but  it  is  better  to  allow  them  to  dry  for  a  few  days. 
One  should  never  use  a  cement  for  sealing  preparations  in  balsam  or 
other  resinous  media  if  the  solvent  of  the  cement  is  also  a  solvent 
of  the  balsam,  etc.  Otherwise  the  cement  will  soften  the  balsam  and 
finally  run  in  and  mix  with  it,  and  partly  or  wholly  ruin  the  prepa- 
ration. Shellac  is  an  excellent  cement  for 
sealing  balsam  preparations,  as  it  never  runs 
in.  Balsam  preparations  are  rarely  sealed. 
§  509.  Example  of  mounting  in  glycerin 
jelly.  —  For  this  select  some  stained  and 
isolated  muscular  fibers  or  other  suitably  pre- 
pared objects  (§  514-519).  Arrange  them  on 
the  middle  of  a  slide,  using  the  centering 
card,  and  mount  in  glycerin  jelly  as  directed 
in  §  507.  Air  bubbles  are  not  easily  removed 
from  glycerin  jelly  preparations,  so  .care 
should  be  taken  to  avoid  them. 

§  510.  Mounting  objects  in  resinous  media. 

-  While  the  media  miscible  with  water  offer 
FIG.  104.    SMALL  SPIRIT  •,  r  i 

LAMP   USED    AS    A    CON-    manv  advantages  for  mounting  animal  and 

TAINER  FOR  GLYCERIN,    vegetable  tissues,  the  preparations  so  made 

liable  to  deteriorate.     In 


BALSAM,  ETC. 


are  liable  to  deteriorate.  In  many  cases, 
also,  they  do  not  produce  sufficient  transparency  to  enable  one  to  use 
high  enough  powers  for  the  demonstration  of  minute  details. 

By  using  sufficient  care  almost  any  tissue  may  be  mounted  in  a 
resinous  medium  and  retain  all  its  details  of  structure. 

For  the  successful  mounting  of  an  object  in  a  resinous  medium  it 
must  in  some  way  be  deprived  of  all  water  and  all  liquids  not  miscible 
with  the  resinous  mounting  medium.  There  are  two  methods  of 
bringing  this  about:  (A)  By  drying  or  desiccation  (§511),  and  (B) 
by  successive  displacements  (§  513). 

§  511.  Order  of  procedure  in  mounting  objects  in  resinous 
media  by  desiccation: 

i.  The  object  suitable  for  the  purpose  (fly's  wings,  etc.)  is  thor- 
oughly dried  in  dry  air  or  by  gentle  heat. 


CH.  X] 


MOUNTING    MICROSCOPIC    OBJECTS 


329 


2.  The  object  is  arranged  as  desired  in  the  center  of  a  clean  slide 
on  the  centering  card  (fig.  192). 

3.  A  drop  of  the  mounting  medium  is  put  directly  upon  the  object 
or  spread  on  a  cover-glass. 

4.  The  cover-glass  is  put  on  the  specimen  with  fine  forceps  (fig. 
190),  but  in  no  case  does  one  breathe  on  the  cover  as  when  media 
miscible  with  water  are  used. 

5.  The  cover-glass  is  pressed  down  gently. 

6.  The  slide  is  labeled. 

7.  The  preparation  is  catalogued  and  safely 
stored  (§  526). 

§  512.  Example  of  mounting  in  balsam  by 
desiccation.  —  Find  a  fresh  fly,  or,  if  in  winter, 
procure  a  dead  one  from  a  window  sill  or  a 
spider's  web.  Remove  the  fly's  wings,  being 
especially  careful  to  keep  them  the  dorsal  side 
up.  With  a  camel's  hair  brush  remove  any 
dirt  that  may  be  clinging  to  them.  Place  a 
clean  side  on  the  centering  card,  then  with 
fine  forceps  put  the  two  wings  within  one  of 
the  guide  rings.  Leave  one  dorsal  side  up, 
turn  the  other  ventral  side  up.  Spread  some 
Canada  balsam  on  the  face  of  the  cover-glass 
and  with  the  fine  forceps  place  the  cover  upon 
the  wings  (fig.  190).  Probably  some  air- 
bubbles  will  appear  in  the  preparation,  but 
if  the  slide  is  put  in  a  warm  place  these  will  soon  disappear.  Label, 
catalogue,  etc. 

§  513.  Mounting  in  resinous  media  by  a  series  of  displacements. 
-  For  examples  of  this  see  the  procedure  in  the  paraffin  and  in  the 
collodion  methods,  Ch.  XL  The  first  step  in  the  series  is  dehydration; 
that  is,  the  water  is  displaced  by  some  liquid  which  is  miscible  both 
with  the  water  and  the  next  liquid  to  be  used.  Strong  alcohol  (95  % 
or  stronger)  is  usually  employed  for  this.  Plenty  of  it  must  be  used 
to  displace  the  last  trace  of  water.  The  tissue  may  be  soaked  in  a 
dish  of  the  alcohol,  or  alcohol  from  a  pipette  may  be  poured  upon  it. 


FIG.  195.  CONTAINER 
FOR  CANADA  BALSAM, 
GLYCERIN  JELLY,  ETC. 

Cover  The  glass  cover 
to  keep  out  dust  and  pre- 
vent evaporation. 

Rod  The  glass  rod  for 
transferring  the  contents 
of  the  container  to  the 
slide. 


330  ISOLATION  OF  TISSUE  ELEMENTS  [Cn.  X 

Dehydration  usually  occurs  in  the  thin  objects  to  be  mounted  in 
balsam  in'  5  to  15  minutes.  If  a  dish  of  alcohol  is  used  it  must  not  be 
used  too  many  times,  as  it  loses  in  strength. 

The  second  step  is  clearing.  That  is,  some  liquid  which  is  miscible 
with  the  alcohol  and  also  with  the  resinous  medium  is  used.  This 
liquid  is  highly  refractive  in  most  cases,  and  consequently  this  step  is 
called  clearing  and  the  liquid  a  clearer.  The  clearer  displaces  the  alco- 
hol, and  renders  the  object  more  or  less  translucent.  In  case  the 
water  was  not  all  removed,  a  cloudiness  will  appear  in  parts  or  over 
the  whole  of  the  preparation.  In  this  case  the  preparation  must  be 
returned  to  alcohol  to  complete  the  dehydration. 

One  can  tell  when  a  specimen  is  properly  cleared  by  holding  it  over 
some  dark  object.  If  it  is  cleared  it  can  be  seen  only  with  difficulty, 
as  but  little  light  is  reflected  from  it.  If  it  is  held  toward  the  window, 
however,  it  will  appear  translucent. 

The  third  and  final  step  is  the  displacement  of  the  clearer  by  the 
resinous  mounting  medium. 

The  specimen  is  drained  of  clearer  and  allowed  to  stand  for  a  short 
time  till  there  appears  the  first  sign  of  dullness  from  evaporation  of 
the  clearer  from  the  surface.  Then  a  drop  of  the  resinous  medium 
is  put  on  the  object,  and  finally  a  cover-glass  is  placed  over  it,  or  a 
drop  of  the  mounting  medium  is  spread  on  the  cover  and  it  is  then  put 
on  the  object.  For  abundant  examples  see  the  next  chapter. 

ISOLATION  or  HISTOLOGIC  ELEMENTS 

§  514.  Isolation,  general.  —  For  a  correct  conception  of  the  forms 
of  the  cells. and  fibers  of  the  various  organs  of  the  body,  one  must  see 
these  elements  isolated  and  thus  be  able  to  inspect  them  from  all  sides. 
It  frequently  occurs  also  that  the  isolation  is  not  quite  complete,  and 
one  can  see  in  the  clearest  manner  the  relations  of  the  cells  or  fibers 
to  one  another. 

The  chemical  agents  or  solutions  for  isolating  are,  in  general,  the 
same  as  those  used  for  hardening  and  fixing.  But  the  solutions  are 
only  about  one-tenth  as  strong  as  for  fixing,  and  the  action  is  very 
much  shorter,  that  is,  from  one  or  two  hours  to  as  many  days.  In 
the  weak  solution  the  cell  cement  or  connective  tissue  is  softened  so 


CH.  XI 


ISOLATION  OF  TISSUE  ELEMENTS 


331 


that  the  cells  and  fibers  may  be  separated  from  one  another,  and  at 
the  same  time  the  cells  are  preserved.  In  fixing  and  hardening,  on 
the  other  hand,  the  cell  cement,  like  the  other  parts  of  the  tissue,  is 
made  firmer.  In  preparing  the  isolating  solutions  it  is  better  to 


FIG.    196,    197.     SHELL  VIAL   AND   COMSTOCK,   BENT-NECK  SPECIMEN  BOTTLE. 

FIG.  196.  Shell  vial  with  turned  lip.  One  can  have  almost  any  size  and  length 
desired.  Those  of  22  x  65  mm.  and  30  X  90  mm.  have  been  found  most  useful. 
The  larger  ones  are  excellent  for  staining  single  slides  or  pairs. 

FIG.  197.  The  Comstock,  bent-neck  specimen  bottle  is  very  useful  for  keeping 
small  animals  straight. 

dilute  the  fixing  agents  with  normal  salt  solution  than  merely  with 
water  (§  584). 

§  515.  Example  of  isolation.  —  Place  a  piece  of  the  trachea  of  a 
very  recently  killed  animal,  or  the  roof  of  a  frog's  mouth,  in  formalde- 
hyde dissociator  in  a  shell  vial  or  glass  box.  After  half  an  hour,  up  to 
two  or  three  days,  excellent  preparations  of  ciliated  cells  may  be 
obtained  by  scraping  the  trachea  or  roof  of  the  mouth  and  mounting 
the  scrapings  on  a  slide.  If  one  proceeds  after  one  hour,  probably 
most  of  the  cells  will  cling  together,  and  in  the  various  clumps  will 
appear  cells  on  end  showing  the  cilia  or  the  bases  of  the  cells,  and 
other  clumps  will  show  the  cells  in  profile.  By  tapping  the  cover 


332 


ISOLATION  OF  TISSUE  ELEMENTS 


[CH.  X 


ooo 
ooo 
ooo 
ooo 
ooo 


gently  with  a  needle  holder  or  other  light  object  the  cells  will  separate 
from  one  another,  and  many  fully  isolated  cells  will  be  seen. 

§  516.  Isolation  by  means  of  formaldehyde.  —  Formaldehyde  in 
normal  salt  solution  is  one  of  the  very  best  dissociating  agents  for 
brain  tissue  and  all  the  forms  of  epithelium.  It  is  prepared  as  follows: 
2  cc.  of  formal  (that  is,  a  40  %  solution  of  formaldehyde)  are  mixed 
with  1000  cc.,  of  normal  salt  solution.  This  acts  quickly  and  pre- 
serves delicate  structures  like  the  cilia  of  ordi- 
nary epithelia  and  also  of  the  endymal  cells  of 
the  brain.  It  is  satisfactory  for  isolating  the 
nerve  cells  of  the  brain.  For  the  epithelium  of 
the  trachea,  intestines,  etc.,  the  action  is  suffi- 
cient in  half  an  hour;  good  preparations  may 
also  be  obtained  any  time  within  two  days  or 
more.  The  action  on  nerve  tissue  of  the  brain 
and  myel  or  spinal  cord  is  about  as  rapid. 

§  517.  Staining  the  cells.  —  Almost  any  stain 
may  be  used  for  the  formalin  dissociated  cells. 
For  example,  one  may  use  eosin.  This  may  be 
drawn  under  the  cover  of  the  already  mounted 
preparation  (fig.  193),  or  a  new  preparation 
may  be  made  and  the  scrapings  mixed  with  a 
drop  of  eosin  before  putting  on  the  cover-glass. 
It  is  an  advantage  to  study  unstained  prepara- 
tions, otherwise  one  might  obtain  the  erroneous 
opinion  that  the  structure  cannot  be  seen  unless 

it  is  stained.  The  stain  makes  the  structural  features  somewhat 
plainer;  it  also  accentuates  some  features  and  does  not  affect  others 
so  markedly.  Congo  red  is  excellent  for  most  isolated  cells. 

§  518.  Permanent  preparations  of  isolated  cells.  —  If  one  desires 
to  make  a  permanent  preparation  of  isolated  cells  it  may  be  done  by 
placing  a  drop  of  glycerin  at  the  edge  of  the  cover  and  allowing  it 
to  diffuse  under  the  cover,  or  the  diffusion  may  be  hurried  by  using 
a  piece  of  blotting  paper,  as  shown  in  fig.  193.  One  may  also  make 
a  new  preparation  by  mixing  thoroughly  some  of  the  isolated  material 
with  congo-glycerin.  After  a  few  minutes  the  cover-glass  may  be 


FIG.  198.  BLOCK  WITH 
HOLES  FOR  SHELL- 
VIALS. 

The  blocks  are  about 
33  mm.  thick  and  the 
holes  are  bored  clear 
through,  then  a  board 
about  5  mm.  thick  is 
nailed  on  the  bottom. 


CH.  X] 


ISOLATION    OF    TISSUE    ELEMENTS 


333 


put  on  and  sealed  (§  508).  If  one  adds  congo-glycerin  to  a  consid- 
erable amount  of  the  isolated  material  it  may  be  kept  and  used 
at  any  time. 

§  519.  Isolation  of  muscular  fibers.  —  For  this  the  formal  disso- 
ciator  may  be  used  (§516),  but  the  nitric  acid  method  is  more  suc- 
cessful (§  560).  The  fresh  muscle  is  placed  in  this  in  a  glass  vessel. 
At  the  ordinary  temperature  of  a  sitting  room  (20  degrees  centigrade) 


FIG.  199.    MOIST  CHAMBER  AND  MOIST  PREPARATIONS. 

A  Bowl  (B)  inverted  over  a  plate  .(P)  containing  water  and  a  glass  shelf 
supported  on  glass  rods.  The  slides  (S)  are  supported  on  the  glass  shelf.  This 
makes  a  very  efficient  and  cheap  moist  chamber. 

B  Cover-glasses  (C)  made  slightly  eccentric  and  containing  between  them 
the  object  to  be  kept  moist.  By  using  cover-glasses  the  specimen  can  be  examined 
from  both  sides,  and  as  part  usually  remains  with  each  cover-glass,  two  perma- 
nent preparations  can  be  made. 

C  Slide  (S)  with  a  cover-glass  (C)  extending  slightly  over  one  edge  so  that  it 
can  be  lifted  up  without  danger  of  sliding  it  along  and  thus  disarranging  the 
specimen. 

the  connective  tissue  will  be  so  far  gelatinized  in  from  one  to  three 
days  that  it  is  easy  to  separate  the  fascicles  and  fibers  either  with 
needles  or  by  shaking  in  a  test  tube  or  shell  vial  with  water.  It 
takes  longer  for  some  muscles  to  dissociate  than  others,  even  at  the 
same  temperature,  so  one  must  try  occasionally  to  see  if  the  action 
is  sufficient.  When  it  is,  the  acid  is  poured  off  and  the  muscles  washed 
gently  with  water  to  remove  the  acid.  If  one  is  ready  to  make  the 
preparations  at  once  they  may  be  isolated  and  mounted  in  water. 
If  it  is  desired  to  keep  the  specimen  indefinitely  or  several  days,  the 
water  should  be  poured  off  and  2  %  formaldehyde  added.  The  speci- 


334 


MICROSCOPIC  ANIMALS  AND  PLANTS 


[CH.  X 


A  B 

FIG.  200.  PIPETTES 
FOR  LIQUIDS  AND  FOR 
SPECIMENS. 

A  Pipette  for  liquids. 
This  is  about  one-third 
size. 

B  Pipette  for  hand- 
ling ova  and  other  deli- 
cate specimens. 

/  The  rubber  bulb 
tied  to  the  glass  part. 
It  is  about  natural  size. 

2  Glass  rod.     The 
upper  end  is  fluted  so 
that   the  rubber  bulb 
will  not  come  off,  and 
the  lower  end  is  care- 
fully smoothed  by  heat- 
ing.    To  prevent  small 
ova  and  other  objects 
getting  into  the  bulb, 
some  fine  gauze  may  be 
tied  over  the  upper  end. 

3  Soft  rubber  tube 
over    the    lower    end. 
This  is  not  absolutely 
necessary,  but  the  soft 
rubber  is  less  liable  to 
injure  delicate  objects 
than  the  hard  glass. 


mens  may  be  mounted  in  glycerin,  glycerin  jelly, 
or  balsam.  Glycerin  jelly  is  the  most  satisfac- 
tory, however. 

COLLECTION  AND  STUDY  OF  MICROSCOPIC 
ANIMALS  AND  PLANTS 

§  520.  Collection  of  material.  —  There  are 
many  microscopic  forms  in  nature  that  need  no 
other  preparation  than  mounting  on  a  glass 
slide.  If  low  powers  are  used  a  cover-glass 
may  be  omitted,  but  if  high  powers  are  to  be 
used  a  cover-glass  must  be  put  over  the  object 
to  protect  the  objective  as  well  as  the  object, 
and  to  make  the  optical  corrections  of  the  ob- 
jective perfect  (§  460). 

The  easiest  places  to  find  things  most  in- 
teresting and  beautiful  is  in  the  water  of  pools 
and  along  the  shores  of  streams  where  the  water 
is  quiet.  Go  to  some  pond  or  stream  and  along 
the  shore  where  it  is  shallow ;  take  some  of  the 
vegetation  and  the  mud,  put  in  a  pail  or  dish, 
and  take  to  the  home  or  laboratory.  Put  the 
water  and  vegetation  in  a  plate  or  other  shal- 
low vessel  and  put  it  in  about  the  same  light 
that  it  had  in  nature.  In  a  few  hours,  when  the 
mud  has  settled  the  conditions  will  be  nearly  as 
in  nature,  and  by  the  use  of  fine  forceps  or  one 
of  the  pipettes  (fig.  190,  200),  gather  some  of 
the  water  with  scrapings  from  some  of  the  vege- 
tation, or  some  of  the  water  and  mud.  Put  it 
on  a  slide,  cover  and  examine.  There  may  be 
much  to  see  or  very  little.  One  must  persevere 
and  finally  there  will  come  a  kind  of  instinctive 
knowledge  where  to  find  things.  It  is  also  a 
good  plan  to  use  the  tripod  or  other  magnifier 
and  examine  the  dish.  Often  much  can  be  seen 
in  that  way,  and  one  will  get  a  hint  where  to  col- 


CH.  X] 


MICROSCOPIC  ANIMALS  AND  PLANTS 


335 


FIG.  201.    TRIPOD  MAGNIFIER. 


lect  the  bits  to  put  on  the  slide  for  examination.     Do  not  use  distilled 
water  for  these  organisms,  but  water  from  the  source  of  supply. 

(For  food  see  §  521).  i i _^ 

§  521.  Infusoria  and  bacteria;  In- 
fusions. —  One  of  the  best  ways  to 
get  a  large  variety  of  living  forms,  ani- 
mal and  vegetable,  is  to  make  such  a 
gathering  as  described  above  and  to 
put  it  into  a  small  fruit  jar  or  other 
wide  open  vessel,  and  to  put  with  it 
some  of  the  stems  of  the  grass  along 
the  stream.  If  in  a  moderately  warm 
place  for  a  day  or  more  this  collection 

will  be  found  swarming   with   living   things.     Soon,  however,  the 

numbers  will  lessen  and  finally  there 
will  be  very  few  left.  These  living 
things  need  food.  One  of  the  good 
foods  for  them  is  the  soup  made  from 
boiling  up  some  of  the  grass  and  hay 
found  near  the  natural  habitat.  Any 
good  hay  may  be  used,  however.  When 
the  soup  is  cool  add  some  of  it  to  the 
vessel  containing  the  organisms,  or 
what  is  better  take  another  dish,  add 
the  soup  and  a  fair  amount  of  the 
liquid  from  the  first  gathering.  Usu- 
ally this  new  supply  will  be  as  rich  in 
MAGNIFIER  SUP-  life  as  was  the  original  gathering.  (See 
under  Neutral  Red  (§  582)  for  experi- 
ment in  staining  live  forms.) 

§  522.   Diatoms.  —  These  are  plants 


FIG.    202. 
PORTED  BY  A  FOCUSING,  JOINTED 
HOLDER. 


Base    The  heavy  iron  base  to 
keep  the  apparatus  steady. 


Rack    and    pinion    for  with  siiicious  shells,  and  are  found  in 
focusing  the  magnifier. 

/  /  -Joints  to  make  it  possible  natural  waters  both  salt  and  fresh.     If 

to  put  the  lens  in  any  desired  one  gQes  j-Q  a  pOncJ  Or  stream  in  May 

or  June  or  July  especially,  the  diatoms 

are  very  abundant.     They  may  be  found  at  any  time,  but  in  the 
spring  most  abundantly,  as  with  most  living  things.     The  brownish 


336  MICROSCOPIC  ANIMALS  AND  PLANTS  [Cn.  X 

or  rusty  looking  substance  on  plants,  rocks,  etc.,  practically  always 
contain  diatoms,  and  sometimes  is  made  up  mostly  of  them.  It  is  most 
interesting  to  study  the  diatoms  alive  and  watch  them  glide  around 
in  the  water.  The  shells  of  the  diatoms  have  been  favorite  objects 
of  study  for  a  long  time.  They  are  often  beautifully  marked.  Being 
silicious,  they  resist  acids,  and  the  living  substance  in  and  around  them 
can  be  destroyed  without  hurting  the  shells.  This  may  be  done  by 
placing  the  material  containing  a  large  number  of  diatoms  in  a  test 
tube  and  when  the  diatoms  have  settled  pour  off  a  part  of  the  liquid 
or  draw  it  out  with  the  pipette  (fig.  200  A),  and  add  an  equal  amount 
of  nitric  acid.  Boil  for  a  few  minutes,  let  the  diatoms  settle,  pour 
off  or  draw  off  ntost  of  the  liquid,  and  add  more  nitric  acid  and  boil 
again.  Finally,  add  water  and  gradually  wash  the  diatom  shells  by 
drawing  off  the  water  and  adding  fresh.  The  shells  should  be  clean 
and  almost  colorless  and  show  their  markings  well.  One  can  take  a 
sample  and  see  if  the  cleaning  is  sufficient.  (For  full  and  elaborate 
directions  see  Boyer's  Diatomaceae  of  Philadelphia  and  Vicinity, 
p.  122-123). 

§  523.  Arranging  minute  objects.  —  Minute  objects  like  diatoms 
or  the  scales  of  insects  may  be  arranged  in  geometrical  figures  or  in 
some  fanciful  way,  either  for  ornament  or  more  satisfactory  study. 
To  do  this  the  cover-glass  is  placed  over  the  guide.  This  guide  for 
geometrical  figures  may  be  a  net-micrometer  or  a  series  of  concen- 
tric circles.  In  order  that  the  objects  may  remain  in  place,  however, 
they  must  be  fastened  to  the  cover-glass.  As  an  adhesive  substance, 
mucilage  or  liquid  gelatin  (§  578),  thinned  with  an  equal  volume  of 
50  %  acetic  acid,  answers  well.  A  very  thin  coating  of  this  is  spread 
on  the  cover  with  a  needle,  or  in  some  other  way,  and  allowed  to  dry. 
The  objects  are  then  placed  on  the  gelatinized  side  of  the  cover  and 
carefully  got  into  position  with  a  mechanical  finger,  made  by  fastening 
a  cat's  whisker  in  a  needle  holder.  For  most  of  these  objects  a  simple 
microscope  with  stand  (fig.  201-202)  will  be  found  of  great  advantage. 
After  the  objects  are  arranged,  one  breathes  very  gently  on  the 
cover-glass  to  soften  the  mucilage  or  gelatin.  It  is  then  allowed  to 
dry,  and  if  a  suitable  amount  of  gelatin  has  been  used  and  it  has  been 
properly  moistened,  the  objects  will  be  found  firmly  anchored.  In 


CH.  X]  LABELING  AND  CATALOGUING  SLIDES  337 

mounting  one  may  use  Canada  balsam  or  mount  dry  on  a  cell  (§  504, 
511).  See  Newcomer,  Amer.  Micr.  Soc.'s  Proc.,  1886,  p.  128;  see 
also  E.  H.  Griffith  and  H.  L.  Smith,  Amer.  Jour,  of  Micros.,  iv,  102, 
v,  87;  Amer.  Monthly  Micr.  Jour.,  i,  66,  107,  113;  Cunningham, 
The  Microscope,  viii,  1888,  p.  237. 

LABELING,  CATALOGUING  AND  STORING  MICROSCOPIC  PREPARATIONS 

§  524.  Every  person  possessing  a  microscopic  preparation  is  inter- 
ested in  its  proper  management; '  but  it  is  especially  to  the  teacher  and 
investigator  that  the  labeling,  cataloguing,  and  storing  of  microscopic 
preparations  are  of  importance.  "To  the  investigator,  his  specimens 
are  the  most  precious  of  his  possessions,  for  they  contain  the  facts 
which  he  tries  to  interpret,  and  they  remain  the  same  while  his  knowl- 
edge, and  hence  his  power  of  interpretation,  increase.  They  thus 
form  the  basis  of  further  or  more  correct  knowledge;  but  in  order  to 
be  safe  guides  for  the  student,  teacher,  or  investigator,  it  seems  to  the 
writer  that  every  preparation  should  possess  two  things:  viz.  a  label 
and  a  catalogue  or  history.  This  catalogue  should  indicate  all  that 
is  known  of  a  specimen  at  the  time  of  its  preparation,  and  all  of  the 
processes  by  which  it  is  treated.  It  is  only  by  the  possession  of  such 
a  complete  knowledge  of  the  entire  history  of  a  preparation  that  one 
is  able  to  judge  with  certainty  of  the  comparative  excellence  of 
methods,  and  thus  to  discard  or  improve  those  which  are  defective. 
The  teacher,  as  well  as  the  investigator,  should  have  this  information 
in  an  accessible  form,  so  that  not  only  he,  but  his  students,  can  obtain 
at  any  time  all  necessary  information  concerning  the  preparations 
which  serve  him  as  illustrations  and  them  as  examples." 

§  525.  Labeling  ordinary  microscopic  preparations.  —  The  label 
should  possess  at  least  the  following  information. 

The  number  of  the  preparation,  its  name  and  date  and  the  thick- 
ness of  the  sections  and  of  the  cover-glass. 

§  526.  Cataloguing  preparations.  —  It  is  believed  from  personal 
experience,  and  from  the  experience  of  others,  that  each  preparation 
(each  slide  or  each  series)  should  be  accompanied  by  a  catalogue  con- 
taining at  least  the  information  suggested  in  the  following  formula. 
This  formula  is  very  flexible,  so  that  the  order  may  be  changed,  and 


338 


LABELING  AND  CATALOGUING  SLIDES 


[CH.  X 


numbers  not  applicable  in  a  given  case  may  be  omitted.  With  many 
objects,  especially  embryos  and  small  animals,  the  time  of  fixing 
and  hardening  may  be  months  and  even  years  earlier  than  the  time 
of  imbedding.  So,  too,  an  object  may  be  sectioned  a  long  time  after 
it  was  imbedded,  and  finally  the  sections  may  not  be  mounted  at  the 
time  they  are  cut.  It  would  be  well  in  such  cases  to  give  the  date  of 
fixing  under  2,  and  under  5,  6  and  8  the  dates  at  which  the  operations, 
were  performed  if  they  differ  from  the  original  date  and  from  one 


Jo  JO 

Cc  (y coven 

t 

Jytrvc. 


FIG.  203.    LABEL  FOR  A  MICROSCOPIC  PREPARATION. 

The  specimen  is  the  myel  (spinal  cord)  of  an  Amphioxus  showing  the  dorsal 
and  ventral  nerve  roots,  and  some  nerve  cells  near  the  middle. 

G    A  nerve-cell  with  glycogen. 

In  the  label  €.15  means  that  the  cover-glass  is  0.15  mm.  in  thickness;  and  s  10  p 
means  that  the  section  is  ten  microns  thick.  The  date  at  the  bottom  shows  when 
the  specimen  was  made. 

another.     In  brief,  the  more  that  is  known  about  a  preparation  the 
greater  its  value. 

§  527.   General  formula  for  cataloguing  microscopic  preparations: 

1.  The  general  name  and  source.     Thickness  of  cover-glass  and 
of  section. 

2.  The  number  of  the  preparation  and  the  date  of  obtaining  and 
fixing  the  specimen;   the  name  of  the  preparator. 

3.  The  special  name  of  the  preparation  and  the  common  and  scien- 
tific name  of  the  object  from  which  it  is  derived.     Purpose  of  the 
preparation. 

4.  The  age  and  condition  of  the  object  from  which  the  preparation 
is  derived.     Condition  of  rest  or  activity;   fasting  or  full  fed  at  the 
time  of  death. 

5.  The  chemical   treatment,  —  the  method  of  fixing,  hardening, 
dissociating,  etc.,  and  the  time  required. 


CH.  X]  LABELING  AND  CATALOGUING  SLIDES  339 

6.  The  mechanical   treatment,  —  imbedded,   sectioned,   dissected 
with  needles,  etc.     Date  at  which  done. 

7.  The  staining  agent  or  agents  and  the  time  required  for  staining. 

8.  Dehydrating  and  clearing  agent,  mounting  medium,   cement 
used  for   sealing. 

9.  The    objectives    and    other    accessories    (micro-spectroscope, 
polarizer,  etc.),  for  studying  the  preparation. 

10.  Remarks,  including  references  to  original  papers,  or  to  good 
figures  and  descriptions  in  books. 

§  528.   A  catalogue  card  written  according  to  this  formula: 

1.  Muscular  Fibers  of  Cat;   Cover  0.15  mm.;   Fibers  2o/z  to  40/4 
thick. 

2.  No.  475.    (Drr.  IX)  Oct.  i,  1891.     S.  H.  G.,  Preparator. 

3.  Tendinous  and  intra-muscular  terminations  of  striated  muscular 
fibers  from  the  Sartorius  of  the  cat  (Felis  domestica). 

4.  Cat  eight  months  old,  healthy  and  well  nourished.     Fasting 
and  quiet  for  12  hours. 

5.  Muscle  pinned  on  cork  with  vaselined  pins  and  placed  in  20 
per  cent  nitric  acid  immediately  after  death  by  chloroform.     Left 
36  hours  in  the  acid;    temperature  20°  C.     In  alum  water  (J  sat. 
aq.  sol.)  i  day. 

6.  Fibers  separated  on  the  slide  with  needles,  Oct.  3. 

7.  Stained  5  minutes  with  Delafield's  hematoxylin. 

8.  Dehydrated  with  95%  alcohol  5  minutes,  cleared  5   minutes 
with  carbol- turpentine,  mounted  in  xylene  balsam;  sealed  with  shellac. 

9.  Use  a  1 6  mm.  for  the  general  appearance  of  the  fibers,  then  a 
2  or  3  mm.  objective  for  the  details  of  structure.     Try  the  micro- 
polariscope  (§  421). 

10.  The  nuclei  or  muscle  corpuscles  are  very  large  and  numerous; 
many  of  the  intra-muscular  ends  are  branched.     See  S.  P.   Gage, 
Proc.  Amer.  Micr.  Soc.,  1890,  p.  132;    Ref.  Hand-book  Med.  Sci., 
Vol.  V,  p.  59- 

§  529.  General  remarks  on  catalogues  and  labels.  — It  is  especially 
desirable  that  labels  and  catalogues  shall  be  written  with  some  imper- 
ishable ink.  Some  form  of  water-proof  carbon  ink  is  the  most  avail- 
able and  satisfactory.  The  water-proof  India  ink,  of  Higgins  or 


340  CABINETS  AND  TRAYS  FOR  SPECIMENS  [Cn.  X 

Weber,  answers  well.  For  ordinary  writing  it  should  be  diluted 
with  one-third  its  volume  of  water  and  a  few  drops  of  strong  ammonia 
added. 

If  one  has  a  writing  diamond  it  is  a  good  plan  to  write  a  label  with 
it  on  one  end  of  the  slide.  It  is  best  to  have  the  paper  label  also,  as  it 
can  be  more  easily  read. 

The  author  has  found  stiff  cards,  12^  x  yj  cm.,  like  those  used  for 
cataloguing  books  in  public  libraries,  the  most  desirable  form  of  cata- 
logue. A  specimen  that  is  for  any  cause  discarded  has  its  catalogue 
card  destroyed  or  stored  apart  from  the  regular  catalogue.  New  cards 
may  then  be  added  in  alphabetical  order  as  the  preparations  are  made. 
In  fact  a  catalogue  on  cards  has  all  the  flexibility  and  advantage 
of  the  slip  system  of  notes. 

Some  workers  prefer  a  book  catalogue.  Very  excellent  book  cata- 
logues have  been  devised  by  Ailing  and  by  Ward  (Jour.  Roy.  Micr. 
Soc.,  1887,  pp.  173,  348;  Amer.  Monthly  Micr.  Jour.,  1890,  p.  91; 
Amer.  Micr.  Soc.  Proc.,  1887,  p.  233). 

The  fourth  division  has  been  added,  as  there  is  coming  to  be  a  strong 
belief,  practically  amounting  to  a  certainty,  that  there  is  a  different 
structural  appearance  in  many  if  not*  all  of  the  tissue  elements,  de- 
pending upon  the  age  of  the  animal,  upon  its  condition  of  rest  or 
fatigue;  and  for  the  cells  of  the  digestive  organs,  whether  the  animal 
is  fasting  or  full  fed.  Indeed  as  physiological  histology  is  recognized 
as  the  only  true  histology,  there  will  be  an  effort  to  determine  exact 
data  concerning  the  animal  from  which  the  tissues  are  derived.  (See 
Minot,  Proc.  Amer.  Assoc.  Adv.  Science,  1800,  pp.  271-289;  Hodge, 
on  nerve  cells  in  rest  and  fatigue,  Jour.  Morph.,  vol.  VII  (1892), 
pp.  95-168;  Jour.  PhysioL,  vol.  XVII,  pp.  129-134;  Gage,  The  Pro- 
cesses of  Life  revealed  by  the  Microscope;  a  Plea  for  Physiological 
Histology,  Proc.  Amer.  Micr.  Soc.,  vol.  XVII  (1895),  pp.  3-29; 
Science,  vol.  II,  Aug.  23,  1895,  pp.  209-218.  Smithsonian  Insti- 
tution, Report  for  1896,  pp.  381-396. 

CABINET  FOR  MICROSCOPIC  PREPARATIONS 

§  530.  While  it  is  desirable  that  microscopic  preparations  should 
be  properly  labeled  and  catalogued,  it  is  equally  important  that  they 


CH.  X] 


CABINETS  AND  TRAYS  FOR  SPECIMENS 


341 


should  be  protected  from  injury.  During  the  last  few  years  several 
forms  of  cabinets  or  slide  holders  have  been  devised.  Some  are  very 
cheap  and  convenient  where  one  has  but  a  few  slides.  For  a  laboratory 
or  for  a  private  collection  where 
the  slides  are  numerous  the  follow- 
ing characters  seem  to  the  writer 
essential: 

(1)  The  cabinet  should  allow  the 
slides  to  lie  flat,  and  exclude  dust 
and  light. 

(2)  Each  slide  or  pair  of  slides 
should  be  in  a  separate  compart- 
ment.   At  each  end  of  the  compart- 
ment should  be  a  groove  or  bevel, 
so  that  upon  depressing  either  end 
of  the  slide  the  other  may  be  easily 
grasped  (fig.  204).     It  is  also  desir- 
able to  have  the  floor  of  the  com- 
partment grooved  so  that  the  slide 
rests  only  on  two  edges,  thus  pre- 
venting soiling   the   slide  opposite 
the  object. 

(3)  Each  compartment  or  each 
space  sufficient  to  contain  one  slide 
of  the  standard  size  should  be  num- 
bered, preferably  at  each  end.     If 
the  compartments  are  made  of  suf- 
ficient width  to  receive  two  slides, 
then  the  double  slides  so  frequently 
used   in   mounting   serial    sections 
may  be  put  into  the  cabinet  in  any 
place  desired. 

(4)  The  drawers  of  the  cabinet  should  be  entirely  independent, 
so  that  any  drawer  may  be  partly  or  wholly  removed  without  dis- 
turbing any  of  the  others. 

(5)  On  the  front  of  each  drawer  should  be  the  number  of  the  drawer 


96 



o 

Jfa.96  /sso 

^e.weLfMcx 

~ 

70 

• 

FIG.  204.  FACE  AND  EDGE  VIEW 
OF  A  CABINET  DRAWER  FOR  MICRO- 
SCOPIC SLIDES. 

96,  70  The  number  of  the  com- 
partment. 

a  b  In  the  compartment  a,  the 
slide  is  resting  in  place  to  show  that 
the  container  touches  the  slide  only 
in  two  places. 

In  b,  the  slide  is  depressed  into 
the  groove  at  one  end  of  the  com- 
partment. It  is  then  easy  to  grasp 
the  slide. 


342 


CABINETS  AND  TRAYS  FOR  SPECIMENS 


[CH.  X 


in  Roman  numerals,  and  the  number  of  the  first  and  last  compart- 
ment in  the  drawer  in  Arabic  numerals  (fig.  205). 

§  531.  Trays  for  slides  and  ribbons  of  sections.  —  Early  in  1897 
the  writer  devised  the  simple  tray  shown  in  fig.  206.  It  was  designed 
especially  for  the  ribbons  of  sections  in  preparing  embryologic  series 

and  for  material  for 
class  work.  As  will 
be  seen  by  the  figure 
the  two  slides  are 
alike  and  the  tray  is 
very  shallow.  It  was 
soon  found  that  the 
wood  forming  the 
bottom  of  the  tray 
was  too  rough  for  rib- 
bons of  sections  and 
smooth  white  paper 
was  put  in  the  tray 
before  the  ribbons 
were  laid  upon  it. 

These  trays  were 
soon  used  for  the 
mounted  prepara- 
tions as  well  as  for 
the  ribbons  of  sec- 
tions. Theywere 
made  of  a  proper  size 
to  fit  the  laboratory 
lockers  (fig.  208)  and  naturally  came  to  be  used  for  storage  instead  of 
the  expensive  slide  cabinets.  For  this  purpose  five  could  be  put  in 
a  single  compartment  of  the  locker  or  thirty-five  in  an  entire  locker. 
As  each  tray  holds  fifty  slides  25  x  75  mm.;  thirty-five  38  x  75  mm., 
and  twenty-five  slides  50  X  75  mm.,  the  saving  of  space  was  very 
great. 

§  532.  Slide  trays  with  tongue,  groove,  and  compartments.  —  In 
the  first  trays  the  edges  were  square  and  sharp.  These  were  rounded 


FIG.  205.     CABINET  FOR  MICROSCOPE  SLIDES. 

This  cabinet  contains  20  drawers  like  that  shown  in 
fig.  204,  and  as  indicated  at  the  right  there  are  420  com- 
partments for  slides. 


CH.  X] 


CABINETS  AND  TRAYS   FOR  SPECIMENS 


343 


in  later  trays,  but  there  still  remained  a  defect,  for  if  one  wished  to 
pile  up  five  to  twenty  trays  on  the  table,  they  would  not  stay  in  an 
even  stack.  To  remedy  this  defect  the  long  way  of  the  frame  was 
tongued  on  one  side  and  grooved  on  the  other,  as  shown  in  fig.  207. 
This  is  a  great  improvement,  as  one  can  make  even  stacks  of  25  or 
50  trays,  and  they  will  stay  in  position.  Furthermore  it  renders  the 
groups  of  five  trays  stored  in  the  locker  compartments  much  easier 


o 

FIG.  206.    SIMPLEST  FORM  OF  SLIDE  TRAY. 

A  Face  view  of  the  slide  tray.  The  screw  eye  at  the  lower  end  is  convenient 
for  pulling  out  a  single  tray. 

B  Sectional  view  of  the  tray  showing  the  thin  board  of  which  it  is  made  and  the 
wooden  frame. 

C     Sectional  view  showing  how  the  frame  is  fastened  to  the  board. 

to  manage,  as  one  can  remove  any  of  the  five  trays  without  getting 
the  others  disarranged,  as  so  often  occurred  with  the  old  form,  lacking 
tongue  and  groove. 

A  defect  of  the  trays  for  storage  is  the  ease  with  which  the  slides 
get  disarranged  unless  the  tray  is  entirely  full.  To  overcome  this 
defect  Mrs.  Gage  divided  one  face  of  the  tray  into  columns  (fig.  207) 
by  means  of  stout  cord  held  in  place  by  using  melted  paraffin  as  a 
cement.  Later  Dr.  Greenman  of  the  Wistar  Institute  divided  one 


344 


CABINETS  AND  TRAYS   FOR  SPECIMENS 


[CH.  X 


face  of  the  tray  into  columns  by  wooden  strips.  This  is  the  best  way. 
With  the  tray  face  in  columns  the  slides  in  a  single  column  may 
become  disarranged,  but  there  is  no  mixing  of  the  slides  of  different 


O 

A 


-ft 


FIG.  207.     SLIDE  TRAY  WITH  COMPARTMENTS,  AND  WITH  TONGUE  AND  GROOVE 
IN  THE  SIDE  PIECES  OF  THE  FRAME. 

A  Face  view  of  the  newest  form  of  slide  tray  showing  the  five  compartments 
and  the  tongue  on  the  side  pieces. 

B  Longitudinal  section  of  the  tray  showing  the  frame  (/)  and  the  partitions, 
i,  2>  3,  4- 

C  Sectional  view  showing  the  side  piece  with  tongue  and  groove  and  the 
method  of  connecting  the  frame  to  the  board.  In  these  new  forms  the  board 
is  not  wood,  but  pulp,  called  beaver-board.  The  partitions  are  of  wood,  and  are 
nailed  in  place,  not  glued. 

columns.  One  side  of  the  tray  remains  smooth  and  can  be  used  for 
ribbons  of  sections  or  for  any  other  purpose  like  the  original  tray 
(fig.  206). 

§  532a.     In  Ithaca,  these  trays  are  manufactured  by  the  H.  J.  Bool  Co.  Inc. 

The  cost  per  100  of  the  original  form  (fig.  206)  is  $45.00.  The  form  with  tongue 
and  groove  sides  costs  $50.00,  and  the  form  with  tongue  and  groove  sides,  one  face 
divided  into  5  spaces  (fig.  207)  is  $60.00  (July,  1920). 


CH.  X] 


CABINETS  AND  TRAYS  FOR  SPECIMENS 


345 


1  i 
J] 

ii 

!> 
i-,1 


* 

== 

r 

— 

- 

•) 

0 

© 

0 

, 

k 

0 

0 

- 

> 

, 

4 


v>- 


-13*"-   -    -        J 


REAGEMT 

ooc 

BOARD! 


-12.'-  H 


000 

coo 

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©oo 


PLANS 
REAGENT  BOAKD5 


• 

KEAGENT  50ARD5  AMD  DRAWERS  ARE 
IHTERCHAMGEABLf    THROUGHOUT,  '' 


tLtVATIOh. 
LOCKERS    Ih    LABORATORIES, 

FIG.  208.     LABORATORY  LOCKEKS  REAGENT  BOARDS  AND  DRAWERS  DESIGNED 

IN    1895. 

(From  the  Journal  of  Applied  Microscopy,  1898,  p.  127). 

The  lockers  designed  in  1899  f°r  Stimson  Hall  are  in  banks  of  12  or  9,  with 
three  vertical  tiers,  not  two  as  shown  in  this  figure.  Everything  is  of  standard 
size  and  hence  completely  interchangeable. 

Measured  over  all,  the  locker  banks  are  329  cm.  high,  and  139.5  cm.  wide  for 
the  large  banks  and  105  wide  for  the  smaller  banks.  Each  individual  locker, 
inside  measure,  is  32  cm.  wide,  70.5  cm.  high,  and  48  cm.  deep.  It  is  divided  by 
7  runs  into  8  compartments.  As  indicated  in  the  sectional  view,  the  entire  space 
may  be  left  free  in  the  locker  or  partly  filled  or  it  may  be  wholly  filled. 

Each  bank  of  lockers  is  lettered,  and  then  the  individual  lockers  numbered  from 
1-12  or  1-9,  the  numbering  is  in  the  order  of  words  in  a  book,  i.e.,  from  left  to 
right.  Of  course  vertical  numbering  is  equally  feasible.  With  this  form  of 
numbering  each  bank  is  practically  independent  and  can  be  changed  in  position 
without  confusion. 


346  REAGENTS  AND  THEIR  PREPARATION  [Cn.  X 

REAGENTS  FOR  MICROSCOPIC  WORK 

§  533.  —  For  much  of  the  work  done  with  a  microscope  the  re- 
agents needed  are  few  and  inexpensive,  but  for  a  large  laboratory 
with  the  diversity  of  investigations  carried  on  the  reagents  are  nu- 
merous, and  some  of  them  expensive.  Below  are  given  some  of  the 
principal  ones  with  the  method  of  their  preparation. 

§  534.  General  on  preparation  of  reagents.  —  In  preparing  re- 
agents both  weights  and  measures  are  used.  As  a  rule  the  amounts 
given  are  those  which  experience  has  shown  to  give  good  results. 
Variations  in  the  proportions  of  the  mixtures  are  sometimes  advan- 
tageous, and  in  almost  every  case  a  slight  change  in  the  proportions 
makes  no  difference.  Most  laboratory  reagents  are  like  food,  good 
even  under  quite  diverse  proportions  and  methods  of  preparation. 
With  a  few,  however,  it  is  necessary  to  have  definite  strengths. 

By  a  saturated  solution  is  meant  one  in  which  the  liquid  has  dissolved 
all  that  it  can  of  the  substance  added.  This  varies  with  the  tempera- 
ture. It  is  well  to  have  an  excess  of  the  substance  present;  then  the 
liquid  will  be  saturated  at  all  temperatures  usually  found  in  the 
laboratory. 

§  535.  Solutions  less  than  10  per  cent.  —  In  making  solutions  where 
dry  substance  is  added  to  a  liquid,  if  the  percentage  is  not  over  10  %, 
the  custom  is  to  take  100  cc.  of  the  liquid  and  add  to  it  the  number  of 
grams  indicated  by  the  per  cent.  That  is,  for  a  5  %  solution  one  would 
take  100  cc.  of  the  liquid  and  5  grams  of  the  dry  substance.  This 
does  not  make  a  strictly  5  %  solution.  For  that  one  should  take  95  cc. 
of  liquid  and  5  grams  of  the  dry  substance;  or,  If  the  percentage  must 
be  exact,  then  one  should  weigh  out  95  grams  of  the  liquid  and  add 
5  grams  of  the  dry  substance. 

§  536.  Solutions  of  10  per  cent  and  more.  —  When  the  percentage 
is  10%  or  over  it  is  better  to  weigh  out  the  number  of  grams  repre- 
senting the  percentage  and  add  to  it  the  right  amount  of  liquid  in 
cubic  centimeters.  For  example,  if  one  were  to  make  a  35%  aqueous 
solution  of  caustic  potash  in  water  then  one  would  add  35  grams  of 
caustic  potash  to  65  cc.  of  water.  If  one  wished  to  make  a  10% 
alcoholic  solution  of  caustic  potash  he  would  add  10  grams  of  caustic 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  347 

potash  to  90  cc.  of  alcohol.  But  here  is  a  case  where  the  alcohol  being 
.of  less  specific  gravity  than  water  the  mixture  would  not  weigh  100 
grams;  and  to  make  the  mixture  weigh  100  grams,  giving  therefore  an 
exact  percentage,  one  should  take  90  grams  of  alcohol  and  add  to  it 
10  grams  of  caustic  potash.  In  practice  in  making  solutions  of  col- 
lodion or  parlodion  one  usually  mixes  ether  and  95%  or  absolute 
alcohol  in  equal  volumes  and  then  for  a  10  %  solution  10  grams  of  the 
dry  soluble  cotton  or  parlodion  are  added  to  90  cc.  of  the  ether-alcohol 
mixture.  But  ether  is  much  lighter  than  water  and  the  alcohol 
somewhat  lighter,  so  that  the  percentage  in  this  case  would  be  more 
than  10%,  because  the  90  cc.  of  alcohol  and  ether  would  weigh  con- 
siderably less  than  90  grams. 

§  537.  Mixtures  of  liquids  to  obtain  a  desired  percentage.  —  It 
frequently  happens  that  it  is  desired  to  obtain  a  lower  percentage  or 
strength  of  a  liquid  than  the  one  in  stock.  This  is  very  readily  done 
according  to  the  general  formula:  Divide  the  percentage  of  the  strong 
solution  by  the  percentage  of  the  desired  solution  and  the  quotient 
will  show  how  many  times  too  strong  the  stock  solution  is. 

To  get  the  desired  strength,  use  i  volume  of  the  strong  stock  solu- 
tion, and  add  to  it  enough  of  the  diluting  liquid  to  make  a  volume 
corresponding  to  the  amount  indicated  by  the  quotient  obtained  by 
dividing  the  percentage  of  the  stock  solution  by  that  of  the  desired 
solution.  For  example,  if  it  is  desired  to  obtain  a  5  %  solution  of 
formaldehyde  from  a  stock  solution  of  40  %  strength,  the  stock  solu- 
tion being  8  times  too  strong,  to  get  the  5  %  solution  i  volume  of 
the  strong  solution  must  be  used  and  7  volumes  of  the  diluting 
liquid  (water).  The  solution  so  obtained  will  be  £  of  the  original 
strength,  or  5%. 

If  a  2  %  solution  were  desired  then  i  volume  of  the  strong  solution 
would  be  taken  and  19  volumes  of  water,  etc. 

§  538.  Mixtures  of  alcohol.  —  For  alcohol  if  one  desires  a  50  % 
solution  it  is  usually  near  enough  correct  to  add  equal  parts  of  95  % 
alcohol  and  water,  but  this  does  not  actually  give  a  50%  solution. 
To  find  the  real  proportions  according  to  the  general  formula:  95  %  -f- 
50%  =  1.9,  i.e.,  for  every  i  cc.  of  95  %  alcohol  should  be  added  0.9  cc. 
of  water  or  for  each  100  cc.  of  95%  alcohol,  90  cc.  of  water.  This 


348 


REAGENTS  AND  THEIR  PREPARATION 


[CH.  X 


even  will  not  give  an  exact  mixture  of  alcohol,  for  a  mixture  of  alcohol 
and  water  diminishes  somewhat  in  volume.  To  get  true  percentages 
an  alcoholometer  for  testing  the  specific  gravity  is  used. 

A  simple  method  of  getting  approximately  correct  mixtures  of 
alcohol  is  the  following:  Pour  the  strong  alcohol  into  a  graduate 
glass  (fig.  2ogAB)  until  the  volume  is  the  same  as  the  desired  per- 
centage; then  add  water  until 
the  volume  is  the  same  as  the 
original  percentage  of  the  alco- 
hol. Example:  To  get  50  %  from 
95  %  alcohol  put  50  cc.  of  95  % 
into  a  graduate  and  fill  the  grad- 
uate to  95  cc.  with  water,  and 
the  resulting  mixture  will  be 
50%  alcohol,  and  so  with  all 
other  strengths.  Here  the 
shrinkage  is  eliminated  from 
consideration,  because  the  water 
and  alcohol  are  not  measured 


separately  and  then  mixed,  but 
one  is  added  to  the  other  until 
a  given  volume  is  attained. 


FIG.  209.    GLASS  GRADUATES  FOR 
MEASURING  LIQUIDS. 


A  Graduate  with  sloping  sides  for 
large  quantities. 

B  Graduate  with  straight  sides  for 
smaller  quantities  and  more  accurate  de- 


termination. 


PREPARATION  or  REAGENTS 

§539.   Albumen   fixative 

(Mayer's).  —  This  consists  of 
equal  parts  of  well-beaten  white  of  egg  and  glycerin.  To  each  50  cc. 
of  this  i  gram  of  salicylate  of  soda  is  added  to  prevent  putrefactive 
changes.  This  must  be  carefully  filtered.  For  method  of  use  see 
Ch.  XI. 

§  540.  Alcohol  (ethyl),  C2H5OH. — Ethyl  or  grain  alcohol  is  mostly 
used  for  histologic  purposes.  (A)  Absolute  alcohol  (i.e.,  alcohol 
of  99%)  is  recommended  for  many  purposes,  but  if  plenty  of 
95%  alcohol  is  used  it  answers  every  purpose  in  histology,  in  a  dry 
climate  or  in  a  warm,  dry  room.  When  it  is  damp,  dehydration  is 
greatly  facilitated  by  the  use  of  absolute  alcohol. 


CH.  X] 


REAGENTS  AND  THEIR  PREPARATION 


349 


(B)  82%  alcohol  made  by  mixing  5  parts  of  95%  alcohol  with  i 
part  of  water. 

(C)  67%  alcohol  made  by  mixing  2  parts  of  95%  alcohol  with  i 
part  of  water.     See  also  §  537-538. 

§  541.  Alcohol  (methyl),  CH5OH.  —  Methyl  alcohol  or  wood  alcohol 
is  much  cheaper  than  ethyl  or  grain  alcohol  on  account  of  the 
revenue  tax  on  ethyl  alcohol.  It  answers  well  for  many  microscopic 


FIG.  210.    GLASS-STOPPERED  BOTTLES  FOR  THE  MORE  USUAL  GRADES  OF  ALCOHOL 
USED  IN  MICROSCOPY. 

purposes.  It  has  been  refined  so  carefully  in  recent  years  that  the 
disagreeable  odor  is  not  very  noticeable. 

§  542.  Denatured  alcohol.  —  This  is  ethyl  or  grain  alcohol  ren- 
dered undrinkable  by  the  addition  of  wood  alcohol  and  benzine  (grain 
alcohol,  89!%;  methyl  alcohol  10%,  and  benzine  i%).  In  some 
cases  the  denaturing  substances  are  somewhat  different,  but  all  render 
the  alcohol  unusable  for  drinking.  It  is  then  free  from  internal 
revenue  tax. 

In  Great  Britain  "Methylated  Spirits"  consists  of  grain  alcohol 
with  10  %  methyl  alcohol.  This  is  used  very  largely  in  microscopic 
work.  In  America  the  addition  of  the  benzine  renders  denatured 
alcohol  also  unfit  for  histological  purposes  if  it  is  to  be  diluted.  The 
addition  of  water  makes  it  milky.  If  methyl  alcohol  alone  or  combined 
with  pyridin  or  some  other  substance  wholly  soluble  in  water  were 
used  as  the  denaturing  substance,  denatured  alcohol  could  be  used 
in  microscopic  work  for  all  the  grades.  That  denatured  as  indicated 
above  can  be  used  only  in  full  strength  or  very  slightly  diluted. 


350  REAGENTS  AND  THEIR  PREPARATION  [CH.  X 

For  educational  and  other  public  institutions  the  U.  S.  government 
grants  the  privilege  of  using  ethyl  alcohol  without  paying  the  revenue 
tax,  but  for  private  institutions  and  for  individuals  it  would  be  a 
great  relief  if  the  denatured  alcohol  could  be  mixed  in  all  proportions 
with  water  without  the  formation  of  precipitates. 

§  543.  Balsam,  Canada  balsam,  balsam  of  fir.  —  This  is  one  of  the 
oldest  and  most  satisfactory  of  the  resinous  media  used  for  mounting 
microscopic  preparations. 

The  natural  balsam  is  most  often  used;  it  has  the  advantage  of 
being  able  to  take  up  a  small  amount  of  water  so  that  if  sections  are 
not  'quite  dehydrated  they  will  clear  up  after  a  time. 

§  544.  Xylene  balsam.  —  This  is  Canada  balsam  diluted  or  thinned 
with  xylene.  It  is  recommended  by  many  to  evaporate  the  natural 
balsam  to  dryness  and  then  to  dissolve  it  in  xylene.  For  some  pur- 
poses, e.g.  for  mounting  glycogen  preparations,  this  is  advantageous; 
but  it  is  unnecessary  for  most  purposes.  Xylene  balsam  requires 
a  very  complete  desiccation  or  dehydration  of  objects  to  be  mounted 
in  it,  for  the  xylene  is  immiscible  with  water. 

The  hydrocarbon,  xylene  (C8Hi0)  is  called  xylol  in  German.  In 
English,  members  of  the  hydrocarbon  series  have  the  termination 
"ene,"  while  members  of  the  alcohol  series  terminate  in  "ol." 

§  545.  Filtering  balsam.  —  Balsam  is  now  furnished  already  filtered 
through  filter  paper.  If  xylene  balsam  is  used  it  may  be  made  thin 
and  filtered  without  heat.  For  filtering  balsam  and  all  resinous  and 
gummy  materials,  the  writer  has  found  a  paper  funnel  the  most  sat- 
isfactory. It  can  be  used  once  and  then  thrown  away.  Such  a 
funnel  may  be  easily  made  by  rolling  a  sheet  of  thick  writing  paper 
in  the  form  of  a  cone  and  cementing  the  paper  where  it  overlaps,  or 
winding  a  string  several  times  around  the  lower  part.  Such  a  funnel 
is  best  used  in  one  of  the  rings  for  holding  funnels,  so  common  in 
chemical  laboratories.  The  filtering  is  most  successfully  done  in  a 
very  warm  place,  like  an  incubator  or  an  incubator  room. 

§  546.  Neutral  balsam.  —  All  the  samples  of  balsam  tested  by 
the  author  have  been  found  slightly  acid.  This  is  an  advantage  for 
carmine  and  acid  fuchsin  stain  or  any  other  acid  stain.  Also  for 
preparations  injected  with  carmine  or  Berlin  blue.  In  these  cases 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  351 

the  color  would  fade  or  diffuse  if  the  medium  were  not  slightly  acid. 
For  hematoxylin  and  many  other  stains  the  acid  is  detrimental.  For 
example,  the  slight  amount  of  acid  in  the  balsam  causes  the  delicate 
stain  in  the  finest  fibers  of  Weigert  preparations  to  fade.  To  neutralize 
the  balsam  add  some  pure  sodium  carbonate,  set  the  balsam  in  a 
warm  place,  and  shake  it  occasionally.  After  a  month  or  so  the  soda 
will  settle  and  the  clear  supernatant  balsam  will  be  found  very  slightly 
alkaline.  Use  this  whenever  an  acid  medium  would  fade  the  stain 
in  the  specimen. 

§  547.  Acid  balsam.  —  As  stated  above  all  balsam  is  naturally 
somewhat  acid,  but  for  various  stains  it  is  desirable  to  increase  the 
acidity.  For  example,  specimens  stained  with  picro-fuchsin,  or 
injected  with  carmine  or  Berlin  blue  are  more  satisfactory  and  last 
longer  with  full  brilliancy  if  the  balsam  is  made  more  acid  than  it 
naturally  is.  For  this  use  10  to  20  drops  of  glacial  acetic  or  formic 
acid  to  100  cc.  of  balsam. 

§  548.  Borax  carmine  for  in  toto  staining.  —  Borax  4  grams; 
carmine  3  grams;  water  100  cc.  Shake  frequently  for  several  days 
and  then  filter  and  add  100  cc.  of  67  %  alcohol.  After  3  to  4  days  it 
may  be  necessary  to  filter  again.  Good  for  in  toto  staining  after  almost 
any  fixer.  Put  the  object  to  be  stained  from  alcohol  into  a  vial  with 
plenty  of  stain.  After  a  day  or  two  change  the  stain.  Stain  4  to 
5  days.  Remove  to  67%  alcohol,  adding  4  drops  of  HC1  to  each 
100  cc.  of  alcohol.  After  one  day  remove  to  82  %  alcohol.  Change 
the  alcohol  till  no  more  color  comes  away,  then  proceed  to  section. 
Remember  that  objects  stained  in  toto  may  be  mounted  directly  in 
balsam  from  deparaffining  xylene. 

§  549.  Carmine  for  mucus  (mucicarmin).  —  One  can  buy  the  dry 
powder  or  preferably  prepare  the  stain.  To  prepare  it  take  i  gram  of 
Carmine  No.  40  and  \  gram  of  pure  dry  ammonium  chlorid.  If  the 
latter  is  slightly  moist,  dry  it  in  an  evaporating  dish  in  a  sand  bath. 
Mix  the  ammonium  chlorid  and  the  carmine  and  add  2  cc.  of  water. 
Mix  well  and  heat  over  a  sand  bath,  constantly  mixing  with  a  glass 
rod.  Continue  the  heating  until  the  carmine  colored  mass  becomes 
very  dark  red.  It  will  take  3  to  10  minutes  for  this.  The  heat 
should  not  be  too  great. 


352  REAGENTS  AND  THEIR  PREPARATION  [Cn.  X 

Dissolve  the  dark  red  mixture  in  100  cc.  of  50  %  alcohol.  For  use, 
dilute  five  or  tenfold  with  tap  water.  This  stains  best  after  mercuric 
fixers.  One  must  not  collodionize  sections  to  be  stained  with  this, 
as  the  carmine  stains  the  collodion  very  deeply.  Stain  the  sections 
first  with  hematoxylin  as  usual;  then  stain  i  to  5  hours  or  longer  with 
the  dilute  mucicarmin.  The  mucus  in  goblet  cells,  in  the  mucous 
part  of  the  salivary  glands,  etc.,  will  be  red.  Nuclei  will  be  stained 
with  hematoxylin.  Mount  in  balsam  (§  513). 

§  550.  Cedar-wood  oil.  —  For  penetrating  tissues  and  preparing 
them  for  infiltration  with  paraffin,  thick  oil  is  recommended  by  Lee. 
For  tissues  fixed  with  osmic  acid  for  fat  the  thick  oil  is  necessary, 
but  for  most  histologic  and  embryologic  work,  that  known  as  Cedar- 
wood  Oil  (Florida)  is  excellent,  also  that  known  as  Cedar-wood  Oil 
(true  Lebanon).  These  forms  are  far  less  expensive  than  the  thick  oil. 
The  tissues  should  be  thoroughly  dehydrated  before  putting  them  into 
cedar-wood  oil,  and  they  should  remain  until  they  are  translucent. 

The  thickened  cedar-wood  oil  used  for  homogeneous  immersion 
should  be  obtained  of  the  manufacturers  of  microscopes;  they  natu- 
rally would  supply  the  kind  suitable  for  the  purpose. 

§  551.  Chloroform  (CHC13).  — This  is  used  as  an  anaesthetic  and 
for  clearing  and  imbedding  where  fats  fixed  with  osmic  acid  are  to 
be  preserved  in  the  tissues.  It  is  also  used  for  hardening  collodion, 
in  collodion  imbedding.  It  is  an  excellent  solvent  of  cedar-wood  oil 
and  is  used  for  cleaning  homogeneous  immersion  fluid  (cedar-oil)  from 
objectives,  condensers  and  microscopic  preparations. 

§  552.  Carbol-xylene  clearer.  —  Vasale  recommends  as  a  clearer, 
xylene  75  cc.,  carbolic  acid  (melted  crystals),  25  cc. 

§  552a.  —  Carbol-xylene  and  eosin.  In  order  to  counterstain  with  eosin  during 
the  clearing  process,  the  carbol-xylene  is  charged  with  eosin  as  follows:  A  saturated 
aqueous  solution  of  eosin  is  prepared,  and  to  it  is  added  with  constant  stirring,  hy- 
drochloric acid  until  there  is  a  good  precipitate.  Filter  through  filter  paper.  Wash 
the  precipitate  with  distilled  water  until  the  water  goes  through  pink.  This  indi- 
cates that  the  acid  is  washed  out.  Dry  the  washed  precipitate.  This  is  soluble  in 
the  carbol-xylene  and  enough  should  be  added  to  make  that  pink.  More  or  less 
can  be  used  depending  on  the  depth  of  the  eosin  stain  desired.  That  can  be  regu- 
lated also  by  the  time  the  sections  are  left  in  the  eosined  clearer.  (Freeborn,  Jour. 
Ap.  Microscopy,  Vol.  Ill,  p.  1058). 

§  553.  Carbol-turpentine  clearer.  —  A  satisfactory  and  generally 
applicable  clearer  is  carbol  turpentine,  made  by  mixing  carbolic  acid 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  353 


crystals  (Acidcum  carbolicum.  A.  phenicum  crystallizatum)  40  cc. 
with  rectified  oil  of  turpentine  (Oleum  terebinthinae  rectificatium) 
60  cc.  If  the  carbolic  acid  does  not  dissolve  in  the  turpentine, 
increase  the  "turpentine,  thus:  carbolic  acid  30  cc.,  turpentine  70  cc. 

This  clearer  is  not  so  good  as  the  preceding  for  mounting  objects 
which  have  been  stained  with  osmic  acid,  as  the  hydrogen  dioxid 
(H2O2)  present  fades  the  blackened  osmic  acid. 

§  554.  Clarifier,  castor-xylene  clarifier.  —  This  is  composed  of  castor 
oil  i  part  and  xylene  3  parts.  (Trans.  Amer.  Micr.  Soc.,  1895,  p.  361.) 
For  the  use  of  this  clarifier,  see  under  the  collodion  method,  (§  635). 

§  555.  Collodion.  —  This  is  a  solution  of  soluble  cotton  or  other 
form  of  pyroxylin  in  equal  parts  of  sulphuric  ether  in  95  %  or  absolute 
alcohol.  In  using  soluble  cotton  for  infiltrating  and  imbedding  tissues 
several  different  strengths  are  used,  commencing  with  weak  and  pro- 
ceeding to  strong  mixtures.  The  last  in  which  the  tissue  is  imbedded 
is  as  thick  a  solution  as  can  be  made.  All  collodion  solutions  should 
be  kept  well  corked,  for  the  ether  and  alcohol  are  very  volatile. 

§555a.  —  The  substance  used  in  preparing  collodion  goes  by  various  names, 
soluble  cotton  or  collodion  cotton  is  perhaps  best.  This  is  cellulose  nitrate,  and 
consists  of  a  mixture  of  cellulose  tetranitrate  C^HieCNOs^Qe  and  cellulose  pentani- 
trate,  C^HjsCNOs^Os.  Besides  the  names  soluble  and  collodion  cotton,  it  is  called 
gun  cotton  and  pyroxylin.  Pyroxylin  is  the  more  general  term  and  includes  several 
of  the  cellulose  nitrates.  Celloidin  is  a  patent  preparation  of  pyroxylin,  more 
expensive  than  soluble  cotton. 

An  American  product  known  as  'iparlodibn  "  has  recently  (1915)  appeared 
to  take  the  place  of  the  celloidin  not  now  obtainable.  It  is  non-explosive,  and 
said  to  be  a  very  pure,  concentrated  form  of  collodion  especially  adapted  to 
the  needs  of  histology  and  embryology.  (Advertising  pages,  Anatomical 
Record,  Dec.,  1915.) 

Soluble  cotton  should  be  kept  in  the  dark  to  avoid  decomposition.  After 
it  is  in  solution  this  decomposition  is  not  so  liable  to  occur.  The  decomposi- 
tion of  the  the  dry  cotton  gives  rise  to  nitrous  acid,  and  hence  it  is  best  to  keep 
it  in  a  box  loosely  covered,  so  that  the  nitrous  acid  may  escape. 

Cellulose  nitrate  is  explosive  under  concussion  and  when  heated  to  150° 
centigrade.  In  the  air,  the  loose  soluble  cotton  burns  without  explosion.  It 
is  said  not  to  injure  the  hand  if  held  upon  it  during  ignition  and  that  it  does 
not  fire  gunpowder  if  burned  upon  it.  So  far  as  known  to  the  writer,  no  acci- 
dent has  ever  occurred  from  the  use  of  soluble  cotton  for  microscopic  pur- 
poses. I  wish  to  express  my  thanks  to  Professor  W.  R.  Orndorff,  organic 
chemist  in  Cornell  University,  for  the  above  information.  (Proc.  Amer.  Micr. 
Soc.,  vol.  XVII  (1895),  pp.  361-370.) 

§  556.  Collodion  for  cementing  sections  to  the  slide.  —  This  is 
a  J  %  solution  made  by  adding  f  gram  of  soluble  cotton  to  50  cc.  of 


354  REAGENTS  AND  THEIR  PREPARATION  [€H.  X 

95  %  or  absolute  alcohol  and  50  cc.  of  sulphuric  ether.  This  may  be 
used  before  deparaffining  or  preferably  afterward.  See  §  622. 

§  557.  Congo  red.  —  Water  100  cc.,  Congo  red  J  gram.  This  is 
a  good  counter  stain  for  hematoxylin. 

§  558.  Congo-glycerin.  —  For  mixing  with  and  staining  isolation 
preparations  (§517)  and  for  a  mounting  medium  this  is  an  excellent 
combination.  It  is  particularly  good  for  nerve  cells. 

§  559.  Decalcifier.  —  For  removing  the  salts  of  lime  from  bone, 
etc.,  one  must  first  fix  and  harden  the  tissue  by  some  approved 
method.  67  %  alcohol  100  cc. ;  strong  nitric  acid  3  cc.  Change  two 
or  three  times.  It  takes  from  3  to  10  days,  depending  on  the  object. 
One  can  tell  when  the  decalcification  is  complete  by  inserting  a  needle. 
If  there  is  no  gritty  feeling  the  work  is  done.  Then  wash  a  few  minutes 
in  water  and  transfer  to  67  %  alcohol.  Then  after  24  hours  use  82% 
alcohol.  It  is  usually  better  to  section  by  the  collodion  method. 
Tissue  is  liable  to  deteriorate  after  being  decalcified,  so  section  it  soon. 

§  560.  Dissociating  Liquids.  —  These  liquids  are  for  preserving 
the  tissue  elements  or  cells  and  for  dissolving  or  softening  the  inter- 
cellular substance  so  that  the  cells  may  be  readily  separated  from 
their  neighbors.  The  separation  is  accomplished  by  (a)  teasing  with 
needles;  (b)  shaking  in  a  liquid  in  a  test  tube;  (c)  scraping  with  a 
scalpel  and  crushing  with  the  flat  of  the  blade ;  (d)  by  tapping  sharply 
on  the  cover-glass  after  the  object  wis  mounted.  One  may  find  it 
desirable  to  use  (d)  with  all  the  methods. 

(1)  Formaldehyde  dissociator. — Strong  formalin  (40%  formalde- 
hyde gas  in  water)   2  cc.     Normal  Salt  solution  1000  cc.     One  can 
begin  work  within  \  hour  and  good  results  may  be  obtained  after  2 
to  3  days  immersion.     Excellent  for  epithelia  and  for  nerve  cells. 

(2)  Miiller's  fluid  dissociator.  —  Miiller's  fluid  i  cc.     Normal  salt 
solution  9  cc.     It  usually  requires  from  i  to  5  days  for  epithelia  to 
dissociate  in  this.     The  action  is  more  rapid  in  a  warm  place. 

(3)  Nitric  acid  dissociator.  — Nitric  acid  20  cc.    Water  80  cc.    This 
is  used  especially  for  muscular  tissue.      It  takes  from  i  to  3  days, 
depending  on  the  temperature.     The  nitric  acid  gelatinizes  the  con- 
nective tissue.      Wash  out  the  acid  with  water  for  a  few  minutes. 
Preserve  in  2%  formaldehyde. 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  355 

§  561.  Elastic  stain.  —  For  staining  elastic  substance  the  resorcin- 
basic-fuchsin-iron-chlorid  of  Weigert  is  available.  The  stain  is 
prepared  as  follows. 

Basic  fuchsin  2  grams.  Resorcin  4  grams.  Water  200  cc.  Boil 
for  several  minutes  (5  to  10).  Add  to  the  boiling  mixture  25  cc.  of  a 
30%  aqueous  solution  of  chlorid  of  iron  (FeCle).  Boil  for  3  to  10 
minutes;  then  add  a  saturated  solution  of  the  iron  chlorid  until  the 
color  is  all  precipitated.  Try  the  liquid  occasionally  by  letting  a  few 
drops  run  down  the  side  of  the  glass  beaker  used  for  the  boiling. 
If  the  color  is  precipitated  it  appears  as  fine  granules  and  the  liquid 
is  almost  uncolored  or  slightly  yellow7. 

Allow  the  liquid  to  cool.  If  there  is  plenty  of  time  let  it  stand  over 
night.  Then  either  pour  off  the  supernatant  liquid  or  if  the  precipi- 
tate has  not  settled  filter  through  filter  paper.  Then  either  scrape 
off  the  precipitate  from  the  filter  paper  or  cut  off  the  lower  end  of  the 
filter  containing  the  precipitate  and  put  it  in  the  beaker.  Add  200  cc. 
of  95  %  alcohol  and  heat  over  a  water  bath  till  the  alcohol  boils. 
Continue  the  boiling  5  minutes  or  more  and  stir  up  the  filter  paper 
so  that  all  the  precipitate  may  be  dissolved.  After  boiling  5  minutes 
or  more  filter  the  hot  alcoholic  solution  into  a  warmed  bottle.  After 
this  alcoholic  solution  is  cool  add  5  cc.  of  strong  hydrochloric  acid. 

Stain  sections  in  this  solution  i  hour,  sometimes  less.  Wash  off 
the  stain  with  95  %  alcohol. 

This  works  well  on  sections  by  the  paraffin  or  the  collodion  method 
and  for  tissues  hardened  in  any  manner. 

§  562.  Eosin.  —  This  is  used  mostly  as  a  contrast  stain  with  hema- 
toxylin,  which  is  almost  a  purely  nuclear  stain.  It  serves  to  stain 
the  cell-body,  ground  substance,  etc.,  which  would  be  too  transparent 
and  invisible  with  hematoxylin  alone.  If  eosin  is  used  alone  it  gives 
a  decided  color  to  the  tissue  and  thus  aids  in  its  study.  Eosin  is 
used  in  alcoholic  and  in  aqueous  solutions.  A  very  satisfactory  stain 
is  made  as  follows:  50  cc.  of  water  and  50  cc.  of  95%  alcohol  are 
mixed  and  xV  of  a  gram  of  dry  eosin  added.  \  %  aqueous  eosin  is  also 
good. 

§  563.  Eosin  in  95  per  cent  alcohol.  —  For  staining  embryos  and 
tissues  so  that  the  tissue  in  the  ribbons  of  sections  may  be  easily  seen 


356  REAGENTS  AND  THEIR  PREPARATION  [Cn.  X 

a  saturated  solution  of  alcoholic  eosin  is  made.  This  is  also  used  for 
staining  with  methylene  blue  (§  564). 

§564.  Eosin-Methylene  blue. — Alcohol-soluble  eosin,  3  grams; 
95%  alcohol,  500  cc.  Methylene  blue,  pure,  2  grams;  95%  alcohol 
50  cc.;  distilled  water  450  cc.  i%  aqueous  solution  of  caustic 
potash,  5  cc. 

For  staining  with  this,  material  hardened  in  a  mercuric  mixture 
is  best.  Stain  sections  or  smears  i  to  5  minutes  in  the  eosin.  Rinse 
well  in  water.  Stain  i  to  5  minutes  in  the  methylene  blue  solution. 
Rinse  well  in  tap  water.  Dehydrate  quickly  with  absolute  alcohol. 
Clear  in  pure  xylene  and  mount  in  neutral  balsam  (§  546). 

§565.  Ether,  ether-alcohol.  —  Sulphuric  ether  (C2H5)2O  is  meant 
when  ether  is  mentioned  in  this  book.  Wherever  ether-alcohol  is 
mentioned  it  means  a  mixture  of  equal  volumes  of  sulphuric  ether 
and  95%  or  absolute  alcohol,  unless  otherwise  stated. 

§566.  Farrant's  solution. — Take  25  grams  of  clean,  dry  gum 
arabic,  25  cc.  of  a  saturated  aqueous  solution  of  arsenious  acid, 
25  cc.  of  glycerin.  The  gum  arabic  is  soaked  for  several  days  in  the 
arsenic  water,  then  the  glycerin  is  added  and  carefully  mixed  with 
the  dissolved  or  softened  gum  arabic. 

This  medium  retains  air  bubbles  with  great  tenacity.  It  is  much 
easier  to  avoid  than  to  get  rid  of  them  in  mounting. 

§  567.  Flemming's  Fluid.  — Water  19  cc.;  i%  osmic  acid  10  cc.; 
10%  chromic  acid  3  cc.;  glacial  acetic  acid  2  cc.  This  osmic  fixer 
is  good  for  very  small  pieces  —  i  to  5  millimeter  pieces,  thickness  not 
over  2  to  3  mm.  Wash  out  with  water  10  to  24  hours.  Then  67  % 
alcohol.  Also  82%  and  95%. 

§568.  Formaldehyde  (HCHO  or  OCH2).  —  This  is  found  in 
the  market  under  the  name  of  " formalin,"  etc.,  and  consists  of  a 
40  %  solution  of  formaldehyde  gas  in  water. 

For  fixing  tissues  and  embryos  a  5  %  solution  is  good  (formalin 
i  cc.,  water  7  cc.,  §  537).  A  common  fixer  is  10  cc.  formalin,  90  cc. 
water.  This  is  frequently  called  10%  formalin;  it  is,  however,  only 
4  %  formaldehyde. 

Tissues  may  stay  in  this  indefinitely.  Small  pieces  are  fixed 
within  an  hour.  Before  hardening  in  alcohol  and  imbedding,  wash 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  357 

out  the  formalin  in  running  water  half  an  hour,  then  harden  a  day  or 
more  in  67  %  and  82  %  alcohol. 

For  preserving  nitric  acid  dissociated  muscle  a  2%  formaldehyde 
solution  is  good.  (Formalin  i  cc.,  water  19  cc.  §  537.)  See  also 
§  516  (i)  for  the  formaldehyde  dissociator. 

§  569.  Glycerin.  —  (A)  One  should  have  pure  glycerin  for  a 
mounting  medium.  It  needs  no  preparation,  unless  it  contains 
dust,  when  it  should  be  filtered  through  filter  paper  or  absorbent 
cotton. 

To  prepare  objects  for  final  mounting,  glycerin  50  cc.,  water  50  cc., 
forms  a  good  mixture.  For  many  purposes  the  final  mounting  in 
glycerin  is  made  in  an  acid  medium,  viz.,  glycerin  99  cc.,  glacial  acetic 
or  formic  acid,  i  cc. 

By  extreme  care  in  mounting  and  by  occasionally  adding  ,a  fresh 
coat  to  the  sealing  of  the  cover-glass,  glycerin  preparations  last  a 
long  time.  They  are  liable  to  be  disappointing,  however.  In  mount- 
ing in  glycerin  care  should  be  taken  to  avoid  air-bubbles,  as  they  are 
difficult  to  get  rid  of.  A  specimen  need  not  be  discarded,  however, 
unless  the  air-bubbles  are  large  and  numerous.  See  also  Congo 
glycerin  §  517-518. 

§  570.  Glycerin  jelly  for  microscopic  specimens.  —  Soak  25  grams 
of  the  best  dry  gelatin  in  cold  water  in  a  pyrex  or  agateware  dish. 
Allow  the  water  to  remain  until  the  gelatin  is  softened.  It  usually 
takes  about  half  an  hour.  When  softened,  as  may  be  readily  deter- 
mined by  taking  a  little  in  the  fingers,  pour  off  the  superfluous  water 
and  drain  well  to  get  rid  of  all  the  water  that  has  not  been  imbibed 
by  the  gelatin.  Warm  the  softened  gelatin  over  a  water  bath  and  it 
will  melt  in  the  water  it  has  absorbed.  Add  about  5  cc.  of  egg  albu- 
men, white  of  egg;  stir  it  well  and  then  heat  the  gelatin  in  the  water 
bath  for  about  half  an  hour.  Do  not  heat  above  75°  or  80°  C.,  for  if 
the  gelatin  is  heated  too  hot  it  will  be  transformed  into  meta-gelatin 
and  will  not  set  when  cold.  Heat  coagulates  the  albumen  and  it  forms 
a  kind  of  flocculent  precipitate  which  seems  to  gather  all  fine  particles 
of  dust,  etc.,  leaving  the  gelatin  perfectly  clear.  After  the  gelatin  is 
clarified,  filter  through  a  hot  flannel  filter  and  mix  with  an  equal 
volume  of  glycerin  and  5  grams  of  chloral  hydrate  and  shake  thor- 


358  REAGENTS  AND  THEIR  PREPARATION  [CH.  X 

oughly.  If  it  is  allowed  to  remain  in  a  warm  place  (i.e.,  in  a  place 
where  the  gelatin  remains  melted)  the  air-bubbles  will  rise  and  dis- 
appear. 

In  case  the  glycerin  jelly  remains  fluid  or  semi-fluid  at  the  ordinary 
temperature  (i8°-2o°  C.),  the  gelatin  has  either  been  transformed  into 
meta-gelatin  by  too  high  a  temperature  or  it  contains  too  much  water. 
The  amount  of  water  may  be  lessened  by  heating  at  a  moderate  tem- 
perature over  a  water  bath  in  an  open  vessel.  This  is  an  excellent 
mounting  medium.  Air-bubbles  should  be  avoided  in  mounting  as 
they  do  not  disappear. 

§  571.  Glycerin  jelly  for  anatomic  preparations.  —  Specimens 
prepared  by  the  Kaiserling  method  or  other  satisfactory  way  may 
be  permanently  preserved  in  glycerin  jelly  prepared  as  follows:  Best 
clear  gelatin,  200  grams.  Kaiserling's  No.  4  solution,  3000  cc.  (Po- 
tassium acetate,  100  grams;  glycerin,  200  cc.;  water,  1000  cc).  Put 
the  gelatin  in  the  potassium-acetate-glycerin-water  mixture  in  an 
agate  pail  and  heat  over  a  gas  or  other  stove.  Stir.  When  the  tem- 
perature is  about  55°  centigrade  add  the  whites  of  three  eggs  well 
beaten,  and  stir  them  in  vigorously.  Make  markedly  acid  by  acetic 
acid.  Continue  the  heating  until  the  mixture  just  boils,  and  then 
filter  through  filter  paper  into  fruit  jars.  It  is  best  to  put  over  the 
filter  paper  two  thicknesses  of  gauze.  A  piece  of  thymol  in  the  top  of 
each  jar  will  prevent  the  growth  of  fungi,  or  one  can  add  5  %  chloral 
hydrate.  Specimens  are  mounted  in  this  jelly  directly  from  the  No. 
4  Kaiserlings,  or  alcoholic  specimens  can  be  soaked  in  water  an  hour 
or  more  and  then  kept  in  some  of  the  melted  jelly  until  well  soaked; 
then  mount  permanently  in  the  glycerin  jelly.  At  the  time  of  mount- 
ing the  gelatin  is  liquefied  over  a  water  bath,  and  for  every  20  cc.  of 
the  gelatin  used  one  drop  of  strong  formalin  is  added.  This  is  to 
prevent  the  liquification  of  the  gelatin  after  the  specimen  is  mounted. 
Let  the  gelatin  cool  gradually  after  the  specimen  is  in  place,  then 
add  some  melted  gelatin  to  make  the  vessel  over  full  and  slide  a  glass 
cover  on  it.  This  excludes  all  air.  The  cover  may  then  be  sealed 
with  the  clear  gelatin  or  glue  used  for  gluing  wood,  or  the  cement 
used  in  mending  crockery.  Finally,  one  can  seal  with  rubber  cement 
if  desired.  (See  W.  H.  Watters,  N.Y.  Med.  Record,  Dec.  22,  1906.) 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  359 

§572.  Chloral  hematoxylin. — Potash  alum,  4  grams;  distilled 
water  125  cc.;  hematoxylin  crystals  TV  gram.  Boil  5  to  10  minutes 
in  an  agate  or.  pyrex  dish.  After  cooling,  add  3  grams  of  chloral 
hydrate  and  put  into  a  bottle.  This  will  stain  more  rapidly  after  a 
week  or  two  if  the  bottle  is  left  uncorked.  It  takes  from  i  to  5 
minutes  to  stain  sections, — sometimes  a  long  time.  Use  after  any 
method  of  fixation. 

It  may  be  prepared  for  work  at  once  by  the  addition  of  a  small 
amount  of  hydrogen  dioxid  (H2O2). 

If  the  stain  is  too  concentrated  it  may  be  diluted  with  freshly 
distilled  water  or  with  a  mixture  of  water,  alum  and  chloral.  If  the 
stain  is  not  sufficiently  concentrated,  more  hematoxylin  may  be 
added.  (Proc.  Amer.  Micr.  Soc.,  1892,  pp.  125-127.) 

§  573.  Iron  hematoxylin.  -*•  For  this  stain  there  are  three  solu- 
tions: (a)  the  mordant  composed  of  a  2%  aqueous  solution  of  ferric 
alum  (iron-ammonium-persulphate);  (b)  a  0.5%  solution  of  hema- 
toxylin (10%  alcoholic  hematoxylin  5  cc.,  distilled  water  95  cc.); 
(c)  the  differentiating  fluid  is  composed  of  the  ferric  alum  diluted 
several  times. 

The  stain  can  be  used  after  any  fixer,  and  the  steps  are  as  follows: 
(i)  mordant  with  the  ferric  alum  i  to  24  hours;  (2)  rinse  the  speci- 
men 10  to  30  minutes  in  water;  (3)  stain  for  3  to  24  hours  in  the 
hematoxylin;  (4)  differentiate  slowly,  watching  the  effect  under  the 
microscope.  For  this  dip  the  slide  into  the  ferric  alum  in  the  differ- 
entiator for  a  few  seconds  and  then  rinse  with  tap  water.  When 
satisfactory  wash  in  running  water  15  to  60  minutes.  The  mordant 
and  stain  may  be  used  several  times. 

§  574.  Hematein.  —  This  is  used  instead  of  hematoxylin,  as  it  is 
believed  to  give  more  satisfactory  results.  Prepare  as  follows :  Put 
a  5  %  solution  of  potash  alum  in  distilled  water  and  boil  or  leave  in  a 
steam  sterilizer  an  hour  or  two.  While  warm  add  i  per  cent  of  hema- 
tein  dissolved  in  a  small  quantity  of  alcohol.  After  the  fluid  has 
cooled  add  2  grams  of  chloral  for  each  100  cc.  of  solution.  (Freeborn, 
Jour.  Ap.  Micr.  1900,  p.  1056.) 

§  575.  lodin  stain  for  glycogen.  —  lodin  ij  gram;  iodid  of  potas- 
sium 3  grams;  sodium  chlorid  ij  grams;  water  300  cc.  For  very 


36o 


REAGENTS  AND   THEIR  PREPARATION 


[Cn.X 


soluble  glycogen  one  can  use  50%  alcohol  300  cc.  instead  of  water. 
The  iodin  stain  is  the  most  precise  and  differential  for  glycogen. 
Tissues  or  embryos  for  glycogen  are  fixed  and  hardened  in  95  %  or 
absolute  alcohol,  and  sectioned  by  the  paraffin  or  by  the  collodion 
method.  For  permanent  preparations  the  paraffin  method  is  best 


FIG.  211-213.    BOTTLES  FOR  FIXING  AND  PRESERVING  TISSUES. 

Fig.  211.     Wide  mouth  specimen  bottle  with  glass  stopper. 
Fig.  212.     Salt  mouth  bottle  with  glass  stopper. 
Fig.  213.     Glass  jar  with  screw  top. 

(§  623).  In  spreading  the  sections  use  this  iodin,  stain  instead 
of  water.  Glycogen  in  the  sections  stains  a  mahogany  red,  and  the 
stain  remains  for  ten  or  more  years  in  the  spread  paraffin  sections. 
Spread  sections  may  be  stained  or  restained  by  immersing  the  slide 
in  iodin  stain. 

Before  mounting  permanently,  deparaffin  with  xylene,  and  mount 
in  melted  yellow  vaseline.  Press  the  cover  down  gently.  Seal  with 
shellac  or  balsam.  (Gage,  Trans,  Amer.  Micr.  Soc.,  iQp6,  pp.  203-205.) 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  361 

§576.  lodin  in  alcohol. — lodin  10  grams;  95%  alcohol  90  cc. 
This  is  the  strong  stock  solution. 

For  removing  the  pin-like  or  granular  mercuric  crystals  from  sections 
of  objects  fixed  in  any  fixer  containing  mercury,  e.g.  Zenker's  fluid, 
etc.,  take  95%  alcohol  500  cc.  and  the  10%  iodin  solution  5  cc.  In 
some  cases,  where  the  amount  of  mercury  in  the  tissue  is  great,  one 
may  use  10  or  even  15  cc.  of  the  strong  stock  solution.  Rinse  the 
slide  well  in  pure  95  %  alcohol  to  remove  the  iodin  after  all  the  crystals 
have  dissolved  (^  an  hour  or  more). 

For  embryos  and  tissues  fixed  in  a  mercuric  fixer  one  can  add  several 
drops  of  the  stock  solution  to  the  alcohol  containing  the  tissue  and 
then  by  changing  the  alcohol  occasionally  the  mercury  will  be  mostly 
removed  before  sectioning.  It  is  readily  removed  from  the  sections 
as  just  described. 

§  577.  Lamp-black  for  ingestion  by  leucocytes.  —  Lamp-black,  2 
grams;  sodium  chlorid,  i  gram;  gum  acacia  (gum  Arabic),  i  gram; 
distilled  water,  100  cc.  Mix  all  thoroughly  in  a  mortar.  The  gum 
arabic  is  to  aid  in  getting  an  emulsion  of  the  lamp-black.  Filter 
through  one  thickness  of  gauze  and  one  of  lens  paper.  If  for  a  mammal 
sterilize  by  boiling.  If  some  of  this  mixture  is  injected  into  an  animal, 
the  leucocytes  will  ingest  the  carbon  particles.  Carmine  may  be 
used  instead  of  lamp-black,  but  it  is  not  as  good  because  not  so  endur- 
ing as  lamp-black. 

§  578.  Liquid  gelatin.  —  Gelatin  or  clear  glue,  75  to  100  grams; 
glacial  acetic  acid  40  cc.  and  water  160  cc.;  95%  alcohol  100  cc. ; 
glycerin  15  to  30  cc.  Crush  the  glue  and  put  it  into  a  bottle  with  the 
acid,  set  in  a  warm  place,  and  shake  occasionally.  After  three  or 
more  days  add  the  other  ingredients.  This  solution  is  excellent  for 
fastening  paper  to  glass,  wood,  or  paper.  The  brush  must  be  mounted 
in  a  quill  or  wooden  handle.  For  labels,  it  is  best  to  use  linen 
paper  of  moderate  thickness.  This  should  be  coated  with  liquid 
gelatin  and  allowed  to  dry.  The  labels  may  be  cut  of  any  desired 
size  and  attached  by  simply  moistening  them,  as  in  using  postage 
stamps. 

Very  excellent  blank  labels  are  now  furnished  by  dealers  in  micro- 
scopic supplies,  so  that  it  is  unnecessary  to  prepare  them  one's  self, 


362  REAGENTS  AND  THEIR  PREPARATION  [Cn.  X 

except  for  special  purposes.  Those  like  that  shown  in  fig.  203  may 
be  had  for  about  $3  for  10,000. 

§679.  Mercuric  chlorid  (HgCl2). — Mercuric  chlorid  7^  grams; 
sodium  chlorid  i  gram;  water  100  cc.  The  solution  is  facilitated 
by  heating  in  an  agate  dish.  Fix  fresh  tissue  in  this  2  to  24  hours. 
Then  transfer  to  67  %  alcohol  a  day  or  more  and  then  to  82  %  alcohol. 
Tissues  fixed  in  mercuric  chlorid  deteriorate;  hence  It  is  better  to 
imbed  them  soon  after  they  are  fixed.  Crystals  of  mercury  are 
removed  from  the  sections  by  the  use  of  iodized  alcohol  (§576). 

§  580.  Methylene  blue,  alkaline.  —  Methylene  blue  2  grams;  95  % 
or  absolute  alcohol  50  cc. ;  distilled  water  450  cc. ;  i  %  aqueous  caustic 
potash  5  cc.  This  stain  works  best  after  a  fixer  containing  mercuric 
chlorid,  like  Zenker's  fluid  (see  §  563  for  eosin  in  alcohol). 

§  581.  Miiller's  fluid.  —  Potassium  dichromate  2\  grams;  sodium 
sulphate,  i  gram;  water  100  cc.  This  is  one  of  the  oldest  fixers.  It 
must  act  a  long  time,  two  weeks  to  10  or  12  wreeks.  This  longer  time 
is  for  nervous  tissue  to  be  stained  for  the  myelin.  Lately  this  fixer 
has  been  combined  with  mercury  (see  Zenker's  fluid  §  592).  Before 
putting  the  tissue  into  67  %  alcohol  it  is  washed  out  in  running  water 
for  24  hours. 

Miiller's  fluid  10  cc.;  normal  salt  solution  90  cc.  forms  an  excel- 
lent dissociator  for  epithelia,  etc.  (§  514). 

§  582.  Neutral  red.  —  This  is  used  especially  for  staining  living 
animals.  It  is  used  in  very  weak  solutions:  -y1,,  gram  red;  1000  cc. 
of  water.  Put  a  few  cubic  centimeters  of  this  solution  into  the  vessel 
containing  the  live  animal,  or  animals.  Infusoria  stain  quickly,  10 
to  20  minutes  or  less.  Vertebrates  may  require  a  few  days.  Try 
it  on  infusoria  by  adding  a  drop  of  the  red  to  several  drops  of  the 
infusion  containing  the  infusoria.  Be  sure  that  there  are  many 
animals  present.  Watch  them  under  the  microscope  and  the  color 
will  be  seen  appearing  in  the  granules  of  the  infusoria.  Then  one 
may  cover  and  study  with  a  high  power  (see  §  521). 

§  583.  Nitric  acid,  HNO3.  —  This  is  employed  for  dissociation 
(nitric  acid  dissociator:  water  80  cc.,  nitric  acid  20  cc.);  as  a  fixer, 
especially  for  chick  embryos  in  the  early  stages  (water  90  cc. ;  nitric 
acid,  10 cc.),  and  as  a  delcacifier  (nitric acid  30:. ;  67  %  alcohol  100  cc.). 


:n.X] 


REAGENTS  AND  THEIR  PREPARATION 


363 


§  584.  Normal  liquids.  —  A  normal  liquid  or  fluid  is  one  which 
does  not  injure  or  change  a  fresh  tissue  put  into  it.  The  perfect 
normal  fluids  for  the  tissues  of  any  animal  are  the  fluids  of  the  body 
(lymph  and  plasma)  of  the  animal  from  which  the  tissue  is  taken. 
The  lymph  or  serum  of  one  species  of  animal  may  be  far  from  normal 
for  the  tissues  of  another  animal  (see  also  §  499). 

The  commonly  used  artificial  normal  fluid  is  a  solution  of  common 
salt  (sodium  chlorid)  in  water,  the 
strength  varying  from  -^  to  TV  per 
cent.  As  indicated  above,  this  normal 
salt  or  saline  solution  is  emproyed  in 
diluting  dissociating  liquids  (§  499) . 

§  585.  Paraffin  wax.  —  A  histologic 
laboratory  requires  two  grades  of  par- 
affin for  ordinary  work.  These  are  hard 
paraffin,  melting  at  about  54°  centi- 
grade, and  a  softer  paraffin  melting  at 
about  43°  centigrade.  Usually  a  mix- 
ture of  equal  parts  answers  very  well. 
It  is  economical  for  a  laboratory  to  buy 
the  paraffin  wax  in  cases  of  about  100 
kilograms. 

All  paraffin  for  imbedding  and  section- 
ing should  be  filtered  through  two  thick- 
nesses of  filter  paper.  For  this,  use  a 
metal  funnel,  heat  the  paraffin  very  hot  in  a  water  bath,  and  then 
heat  the  funnel  occasionally  with  a  Bunsen  flame.  The  warmer  the 
room  the  easier  it  is  to  filter  the  paraffin. 

Filter  the  paraffin  into  small  porcelain  pitchers.  If  the  paraffin 
oven  has  a  compartment  large  enough,  it  is  well  to  keep  one  of  the 
pitchers  in  the  oven;  then  the  paraffin  remains  melted  and  is  ready 
for  use  at  any  time. 

§  586.  Picric-alcohol.  —  This  is  an  excellent  hardener  and  fixer 
for  almost  all  tissues  and  organs.  It  is  composed  of  500  cc.  of  water 
and  500  cc.  of  95  %  alcohol,  to  which  2  grams  of  picric  acid  have  been 
added.  (It  is  a  J  %  solution  of  picric  acid  in  50  %  alcohol.)  It  acts 


FIG.  214.    SPECIMEN  JAR 
WITH  CLAMP. 


364  REAGENTS  AND  THEIR  PREPARATION  [Cn.  X 

quickly,  in  from  one  to  three  days.      (Proc.  Amer.  Micr.  Soc.,  Vol. 

XII    (1890),   pp.    120-122.) 

§  587.  Picro-fuchsin.  —  10  cc.  of  a  i  %  aqueous  solution  of  acid 
fuchsin;  75  cc.  of  a  saturated  aqueous  solution  of  picric  acid.  Stain 
deeply  with  hematoxylin  first;  then  use  the  picro-fuchsin.  Wash 
off  the  picro-fuchsin  with  distilled  water.  Mount  in  non-neutralized 
balsam,  or  better  in  acid  balsam  (balsam  50  cc.,  glacial  acetic  acid  5 
drops).  If  the  white  connective  tissue  is  not  red  enough,  increase 
the  amount  of  acid  fuchsin. 

§  588.  Shellac  cement.  —  Shellac  cement  for  sealing  preparations 
and  for  making  shallow  cells  is  prepared  by  adding  scale  or  bleached 
shellac  to  95  %  alcohol.  The  bottle  should  be  filled  about  half  full  of 
dry  shellac;  then  enough  95%  alcohol  added  to  fill  the  bottle  nearly 
full.  The  bottle  is  shaken  occasionally  and  then  allowed  to  stand 
until  a  clear  stratum  of  liquid  appears  on  the  top.  This  clear,  super- 
natant liquid  is  then  filtered  through  filter  paper  or  absorbent  cotton, 
using  a  paper  funnel  (§  545),  into  an  open  dish  or  a  wide  mouth  bottle. 
To  every  100  cc.  of  filtered  shellac  2  cc.  of  castor  oil  may  be  added  to 
render  it  less  brittle.  The  filtered  shellac  will  be  too  thin,  and  must 
be  allowed  to  evaporate  till  it  is  of  the  consistency  of  thin  syrup.  It 
is  then  put  into  a  capped  bottle,  and  for  use  into  a  small  spirit  lamp 
(fig.  194).  In  case  the  cement  gets  too  thick  add  a  small  amount  of 
95%  alcohol  or  some  thin  shellac.  The  solution  of  shellac  almost 
always  remains  muddy,  and  in  most  cases  it  takes  a  long  time  for 
the  flocculent  substance  to  settle.  One  can  quickly  obtain  a  clear 
solution  as  follows:  when  the  shellac  has  had  time  to  thoroughly 
dissolve,  i.e.,  in  a  week  or  two  in  a  warm  place,  or  in  less  time  if  the 
bottle  is  frequently  shaken,  a  part  of  the  dissolved  shellac  is  poured 
into  a  bottle  and  about  one-fourth  as  much  gasoline  added  and  the 
two  well  shaken.  After  twenty-four  hours  or  so  the  flocculent,  un- 
dissolved  substance  will  separate  from  the  shellac  solution  and  rise 
with  the  gasoline  to  the  top.  The  clear  solution  may  then  be  siphoned 
off  or  drawn  off  from  the  bottom  if  one  has  an  aspirating  bottle.  (R. 
Hitchcock,  Amer.  Monthly  Micr.  Jour.,  July,  1884,  p.  131.) 

If  one  desires  to  color  the  shellac,  the  addition  of  a  strong  alcoholic 
solution  of  some  of  the  coal  tar  colors  is  good,  but  is  liable  to  dissolve 


CH.  X]  REAGENTS  AND  THEIR  PREPARATION  365 

in  the  mounting  medium  when  shellac  is  used  for  sealing.  A  small 
amount  of  lamp-black  well  rubbed  up  in  very  thin  shellac  and  filtered 
is  good  to  darken  the  shellac. 

§  589.  Silvering.  —  Intercellular  substance  stains  brown  or  black 
with  nitrate  of  silver.  Use  i  or  J%  aqueous  solution  on  fresh 
tissue.  Stain  in  the  silver  for  i  or  2  minutes;  then  expose  to  light  in 
water  till  brown.  Fix  in  82%  alcohol  or  5%  formaldehyde.  One 
may  stain  afterward  with  hematoxylin  for  the  nuclei;  mount  in 
glycerin,  glycerin  jelly,  or  in  balsam. 

§  690.  Sudan  III  for  fat.  —  Sudan  III,  or  azo-benzene-azo-jS-naph- 
thol,  was  introduced  by  Daddi  into  histology  in  1896  (Arch.  Ital.  de 
Biologic,  t.  26,  p.  142),  as  a  specific  stain  for  fat.  As  it  is  soluble  in 
all  forms  of  fat  and  oils  and  in  xylene,  alcohol,  etc.,  it  is  impossible 
to  mount  specimens  in  balsam  after  staining.  As  the  fat  of  tissues  is 
removed  by  the  reagents  used  in  the  paraffin  and  collodion  methods 
(see  Ch.  XI) ,  only  teased,  free-hand,  or  frozen  sectioned  material  fresh 
or  fixed  in  some  non-fat  dissolving  fixer  can  be  used  (Miiller's  fluid 
and  5  %  f ormaldelhyde  are  excellent) .  The  tissues  cut  free-hand  or 
with  the  freezing  microtome  or  teased  can  then  be  stained  with  a 
saturated  alcoholic  solution  of  the  Sudan.  It  stains  all  fat  a  brilliant 
red.  Preparations  can  be  preserved  in  glycerin  or  glycerin  jelly. 
This  s.tain  is  largely  used  in  pathology. 

Daddi  used  the  substance  to  feed  animals  and  thus  to  stain  the  fat 
which  was  laid  down  in  the  body  while  the  Sudan  was  fed. 

The  fat  in  the  body  already  deposited  remains  unstained.  This 
substance  then  serves  to  record  the  deposit  of  fat  in  a  given  period. 
In  1907  Dr.  Oscar  Riddle  fed  Sudan  to  laying  hens,  and  the  fat  in  the 
layers  of  yolk  laid  down  during  the  feeding  was  stained  red  (Science, 
XXVII,  1908,  p.  495).  For  staining  the  yolks  of  hen's  eggs  the  hen 
may  be  fed  doses  of  20  to  25  milligrams  of  the  Sudan.  Eggs  so  colored 
hatch  as  usual,  and  the  chick  in  utilizing  the  colored  yolk  stains  its 
body-fat  pink  (Susanna  P.  Gage). 

§  591.  Table  Black.  —  During  the  last  few  years  an  excellent  method 
of  dying  wood  with  anilin  black  has  been  devised.  This  black  is 
lusterless,  and  it  is  indestructible.  It  can  be  removed  only  by  scrap- 
ing off  the  wood  to  a  point  deeper  than  the  stain  has  penetrated. 


366  REAGENTS   AND   THEIR  PREPARATION  [€H.  X 

It  must  be  applied  to  unwaxed  or  unvarnished  wood.  If  wax 
paint  or  varnish  has  been  used  on  the  tables,  that  must  be  first 
removed  by  the  use  of  caustic  potash  or  soda  or  by  scraping  or  planing. 
Two  solutions  are  needed: 

Solution  A 

Copper  sulphate 125  grams 

Potassium  chlorate  or  permanganate 125  grams 

Water .* 1000  cc. 

Boil  these  ingredients  in  an  iron  kettle  until  they  are  dissolved. 
Apply  two  coats  of  the  hot  solution.  Let  the  first  -coat  dry  before 
applying  the  second. 

Solution  B 

Anilin  oil 120  cc. 

Hydrochloric  acid 180  cc. 

Water    1000  cc. 

Mix  these  in  a  glass  vessel,  putting  in  the  water  first.  Apply  two 
coats  without  heating,  but  allow  the  first  coat  to  dry  before  adding 
the  second. 

When  the  second  coat  is  dry,  sandpaper  the  wood  and  dust  off  the 
excess  chemicals.     Then  wash  the  wood  well  with  water.     When 
dry,  sandpaper  the  surface  and  then  rub 
thoroughly  with  a  mixture  of  equal  parts 
turpentine  and  linseed  oil.     The  wood  may 
appear  a  dirty  green  at  first,  but  it  will  soon 
become  ebony  black.     If  the  excess  chem- 
FIG.  215.    DRYING  RACK    icals  are  not  removed  the  table  will  crock. 

WITH  INCLINED  PEGS  FOR    An  occasional  rubbing  with  linseed  oil  and 
BOTTLES.  .  .  .  ... 

turpentine   or   with   turpentine   alone   will 

clean  the  surface.  This  is  sometimes  called  the  Danish  method, 
Denmark  black  or  finish.  See  Jour.  Ap.  Micr.,  Vol.  I,  p.  145;  Bot. 
Zeit.,  Vol.  54,  p.  326;  Bot.  Gazette,  Vol."  24,  p.  66;  Dr.  P.  A.  Fish, 
Jour.  Ap.  Micr.,  Vol.  VI,  pp.  211-212. 

§592.  Zenker's  fluid.  —  Miiller's  fluid  (§581)  100  cc.;  mercuric 
chlorid  5  grams.  Just  before  using  add  5  cc.  of  glacial  acetic  acid 
to  each  100  cc.  of  the  above.  Fix  fresh  tissue  5  to  24  hours.  Wash 


CH.  X]  REAGENTS  AND   THEIR   PREPARATION  367 

out  with  running  water  24  hours.  Then  place  in  67%  alcohol  i  day 
or  more  and  finally  preserve  in  82  %  alcohol.  Tissue  fixed  in  Zenker's 
has  mercuric  crystals.  They  may  be  removed  from  the  tissue  by 
long  treatment  with  iodin,  or  by  putting  the  slide  bearing  the  sections 
in  iodized  alcohol  for  half  an  hour  or  more. 

This  is  an  excellent  fixer,  combining  the  good  qualities  of  mercuric 
chlorid  and  of  the  chromium  compounds.  Tissues  fixed  with  this 
show  well  the  red  blood  corpuscles.  This  is  called  Kelly's  fluid  if 
the  acetic  acid  is  replaced  by  5%  formalin. 

COLLATERAL  READING  FOR  CHAPTER  X 

LEE,  A.  B.  —  The  Micro tomist's  Vade  Mecum,  7th  ed.,  1913. 

KINGSBURY,  B.  F.  —  Histological  Technique,  1915. 

MANN,  G.  —  Physiological  Histology,  1903. 

EHRLICH,  P.,  ET  AL.  —  Enzyklopaedie  der  Mikroscopischen  Technik,  1910. 

WRIGHT,  SIR  A.  E.  —  Principles  of  Microscopy,  1907. 

CARPENTER-DALLINGER.  —  The  Microscope  and  Its  Revelations,  1901. 

SPITTA,  E.  J.  —  Microscopy,  1907. 

'Anatomical  Record. 

Journal  of  the  Royal  Microscopical  Society. 

Transactions  of  the  American  Microscopical  Society. 

Journal  of  Experimental  Zoology. 

Botanical  Gazette. 

BOYER,  C.  S.  —  The  Diatomaceae  of  Philadelphia  and  Vicinity,  1916. 

DUDLEY  AND  THOMAS.  —  Manual  of  Plant  Histology,  1894. 

CHAMBERLAIN,  C.  J.  —  Methods  in  Plant  Histology,  1916. 

STEVENS,  W.  C.  —  Plant  Anatomy,  1915. 

EWART,  A.  J.  —  Protoplasmic  Streaming  in  Plants,  1903. 

BERNARD,  CLAUDE.  —  Lecons  sur  les  Phenomenes  de  la  Vie  communs  aux  Animaux 
et  aux  Vegetaux.  Two  vols.  1878-1879. 

NEEDHAM  &  LLOYD.  —  The  Life  of  Inland  Waters,  1916.  This  is  a  most  impor- 
tant work  for  all  interested  in  water  forms. 


CHAPTER  XI 

FIXING  AND  THE  PRESERVATION  OF  TISSUES,  ORGANS,  AND 
ENTIRE  ORGANISMS.  IMBEDDING,  SECTIONING,  STAINING, 
AND  MOUNTING  FOR  THE  MICROSCOPE.  SERIAL  SECTIONS. 
MODELS 

§  600.    Apparatus  and  material  for  Chapter  XI. 

1.  Bottles  and  vials  for  specimens  9.    Hones  and  strops  for  sharpen- 
and   tissues  (fig.    196-197,  211-214).  ing  knives. 

2.  Dissecting  instruments.  10.    Slide    trays    or    cylinders    for 

3.  Fixing  agents  and  alcohol.  ribbons  of  sections  (fig.  206-207). 

4.  Washing    apparatus    (fig.    216-  n.    Slides  and  cover-glasses. 
217).  12.    Slide  baskets  and   glass-stop- 

5.  Clearing  agents  and  imbedding  pered  jars  (fig.  231-232). 

material.  13.    Staining  liquids  and  mounting 

6.  Infiltrating  oven  and  spreading       media. 

plate  (fig.  219-220,  226-230).  14.    Modeling  material,  —  wax  and 

7.  -Section  razors  and  knives  (fig.       blotting  paper. 

218,223).  15.    Apparatus  for  drawing  sections 

8.  Microtomes.  for  models  (§  671-681). 

§  601.  Fixation  and  preservation  of  organs  and  tissues.  —  By 
fixing  or  fixation  in  histology  is  meant  the  preparation  of  fresh  tissues, 
organs,  embryos  or  small  adult  animals  usually  by  means  of  some 
chemical  mixture,  called  a  "fixer,"  so  that  the  organ,  etc.,  as  a  whole 
and  the  elements  or  cells  composing  it  shall  retain  as  nearly  as  possible 
the  morphologic  characters  present  during  life.  The  more  perfect 
the  fixer  the  nearer  will  be  the  preservation  of  all  structural  details. 

Unfortunately  no  single  "fixer"  preserves  with  equal  excellence  all 
the  structural  details,  and  therefore  it  is  necessary  to  prepare  the 
fresh  tissue  in  several  different  ways  and  to  make  a  composite  of  the 
structural  appearances  found,  thereby  approximating  the  actual  struc- 
ture present  in  the  living  body.  Changes  are  so  rapid  after  death 
that  the  fixation  should  begin  as  soon  as  possible.  For  the  most 
perfect  fixation  the  living  tissue  must  be  put  into  the  fixer. 

With  one  of  the  larger  animals  where  the  whole  animal  is  to  be 

368 


CH.  XI] 


PREPARATION  OF  TISSUES 


369 


used  for  microscopic  study  it  is  a  great  advantage  to  bring  the  fixer 
in  contact  with  all  parts  of  the  body  quickly,  and  that  is  done  by 


FIG.  216.    WASHING  BOXES  FOR  TISSUES  FIXED  IN  A  LIQUID  CONTAINING  MER- 
CURIC CHLORID. 

(From  the  Journal  of  Applied  Microscopy). 

T  Small  stop  cock  or  pet  cock  in  the  usual  water  faucet  so  that  a  small  stream 
may  be  drawn  without  interfering  with  the  large  faucet. 

Only  the  larger  trays  are  now  used,  the  perforated  inner  tray  being  deep  or 
shallow  as  needed. 

washing  out  the  vascular  system  with  normal  salt  solution  and  then 
filling  the  vascular  system  with  the  fixer.     This  method  of  "fixation 


7 


FIG.  217.    METAL  WASHING  BOXES  FOR  TISSUES  FIXED  IN  A  LIQUID  CONTAIN- 
ING MERCURIC  CHLORID. 

(From  the  Journal  of  Applied  Microscopy). 

The  deeper  box  is  now  used  only  and  depending  on  the  size  of  the  pieces  to  be 
washed  the  shallow  or  the  deep  perforated  trays  and  tissue  baskets  are  used.  The 
deep  tray  serves  for  washing  slides  with  Weigert  and  other  stains  which  must  be 
in  water  a  long  time. 


370  MICROTOMES  AND   SECTION   KNIVES  [Cn.  XI 

by  injection"  is  of  great  importance  in  the  histology  of  animals  which 
are  large  enough  to  inject. 

If  the  animal  is  too  small  for  injection  or  one  wishes  only  a  small 
part  of  a  larger  animal,  then  the  pieces  for  fixation  should  be  small, 
say  one  to  three  cubic  centimeters.  Often  as  for  Flemming's  fluid 
(§  567)  and  for  several  others  it  is  better  to  use  pieces  2  to  5  cubic 
millimeters  in  volume. 

Large,  solid  organs  must  be  cut  into  several  pieces  if  the  whole 
is  needed.  For  hollow  organs  the  cavity  may  be  filled  with  the 
fixer  and  the  organ  placed  in  a  vessel  of  the  same. 

The  amount  of  fixer  should  be  10  to  50  times  that  of  the  piece  of 
tissue. 

Of  the  fixers  given  under  "  Preparation  of  Reagents,"  picric  alcohol, 
formaldehyde,  and  Zenker's  fluid  are  suitable  for  almost  every  tissue 
and  organ.  Formalin  has  the  advantage  of  having  strong  penetra- 
tion; hence  it  preserves  whole  animals  fairly  by  immersing  after  filling 
the  abdominal  and  thoracic  cavities.  Formaldehyde  is  excellent 
where  a  study  of  fat  is  in  question,  and  it  is  much  used  as  a  fixer 
where  frozen  sections  are  desired  (§  609).  Remember  the  necessity 
of  removing  mercury  from  sections  of  tissues  fixed  with  a  mercuric 
fixer  (fig.  216-217). 

§  602.  Mechanical  preparation  of  tissues,  etc.,  for  microscopic 
study.  —  A  limited  number  of  objects  in  nature  are  small  enough 
and  transparent  enough,  and  a  limited  number  of  the  parts  of  higher 
animals  are  suitable  for  microscopic  study  without  mechanical  prepa- 
ration except  merely  mounting  them  on  a  microscopic  slide.  Usu- 
ally the  parts  of  animals  are  so  large  and  so  opaque  that  the  histologic 
elements  or  cells  and  their  arrangement  in  organs  can  only  be  satis- 
factorily studied  with  a  microscope  after  the  tissue,  organ,  etc.,  have 
been  teased  apart  with  needles,  or  sectioned  into  thin  layers. 

MICROTOMES  AND  SECTION  KNIVES 

§  603.  The  older  histologists,  those  who  laid  the  foundations  and 
whose  understanding  of  the  finer  structure  of  the  body  was  in  many 
ways  superior  to  the  knowledge  possessed  by  workers  at  the  present 
time,  did  their  mechanical  preparation  with  needles  and  with  sharp 


CH.  XI]  MICROTOMES  AND   SECTION  KNIVES  371 

knives  held  in  the  hand.  They  dealt  also  with  fresh  tissue  more 
largely  than  we  do  at  the  present  day,  and  learned  also  to  distinguish 
tissues  by  their  structure  rather  than  by  their  artificial  coloration. 

It  was  not,  however,  on  account  of  the  lack  of  elaborate  mechanical 
devices  for  sectioning  and  complicated  staining  methods  of  the  present 
day,  but  because  they  put  intelligence  and  zeal  into  their  work  that 
made  them  so  successful. 

If  the  reader  is  interested  in  the  mechanical  means  for  sectioning 
he  is  referred  to  Dr.  C.  S.  Minot's  papers  on  the  history  of  the  micro- 
tome in  the  Journal  of  Applied  Microscopy,  Vol.  VI,  and  to  Gilbert 
Morgan  Smith's  article  in  the  Transactions  of  the  American  Micro- 
scopical Society,  Vol.  XXXIV,  1915,  on  the  Development  of  Botanical 
Microtechnique,  pp.  71-129,  16  pages  of  bibliography;  18  figures, 
showing  early  microscopes  and  microtomes. 

§604.  Types  of  microtomes. — There  are  two  great  types:  (i) 
The  early  type  in  which  the  preparation  to  be  sectioned  is  held  me- 
chanically and  moved  up  by  a  screw,  the  section  knife  being  held  in 
the  hand  and  moved  across  the  object,  usually  with  a  drawing  motion 
as  in  whittling. 

(2)  The  mechanical  type,  in  which  both  specimen  and  knife  are 
mechanically  held  and  guided,  and  the  operator  simply  supplies 
power  to  the  machine,  or  when  an  electric  motor  is  used  the  operator 
starts  and  stops  the  machine  and  uses  his  hands  in  taking  off  the 
ribbon  as  it  is  cut.  The  ribbon  is  wound  on  a  cylinder  or  cut  inta 
the  proper  lengths  for  the  slide  trays  (fig.  206-207). 

In  the  highest  types  of  the  second  class  —  automatic  microtomes  — 
the  operator  only  needs  to  put  the  knife  and  specimen  in  position 
and  sections  of  any  thickness  and  any  number  may  be  produced  in 
a  short  time.  A  skilled  and  experienced  person  can  get  better  results 
here  as  well  as  with  free-hand  sectioning  or  the  hand  microtome. 
Even  automatic  machines  work  better  for  skilled  workmen. 

In  some  forms  the  knife  of  these  automatic  microtomes  is  fixed 
in  position  and  the  object  to  be  sectioned  moves,  while  in  other 
forms  the  object  to  be  sectioned  remains  fixed  and  the  knife  moves. 
Furthermore,  for  sectioning  paraffin,  the'  knife  meets  the  object 
like  a  plane  (straight  cut),  while  for  collodion  sectioning  the  knife 


372  MICROTOMES  AND   SECTION   KNIVES  [Cn.  XI 

is  set  obliquely  and  there  results  an  oblique  or  drawing  cut,  as  in 
whittling. 

§  605.  Section  knives.  —  A  section  knife  should  have  the  following 
characters,  (i)  The  steel  should  be  good.  (2)  The  blade  should  be 
slightly  hollow  ground  on  both  sides.  Why  some  makers  persist  in 
grinding  one  side  flat  is  a  mystery.  (3)  The  edge  of  the  knife  should 
be  straight,  not  curved  as  in  a  shaving  razor.  (4)  The  back  should 
be  parallel  with  the  edge.  (5)  The  blade  should  be  long,  12  to  15 
centimeters,  as  it  takes  no  more  time  or  skill  to  sharpen  a  large  than 
a  small  knife.  (6)  The  blade  should  be  heavy.  There  was  formerly 
a  fashion  of  making  very  thin-bladed  section  knives,  but  that  is  a 


FIG.  218.    SECTION  RAZOR  WITH  HEAVY  BLADE  HAVING  STRAIGHT  BACK 

AND  EDGE. 

great  mistake,  for  the  thin  blade  bends  and  vibrates  in  cutting  firm 
tissue  and  large  pieces.  There  is  no  possible  advantage  in  a  thin- 
bladed  section  knife  for  microtome  work,  but  much  disadvantage 
from  the  lack  of  rigidity.  (See  the  catalogues  of  microtomes  and  sec- 
tion knives  by  the  Bausch  &  Lomb  Optical  Co.  and  the  Spencer 
Lens  Co.) 

§606.  Sharpening  section  knives;  hones  and  strops. — Perhaps 
it  should  be  taken  for  granted  that  any  one  would  appreciate  the 
impossibility  of  making  good  sections  with  a  dull  section  knife,  but 
experience  teaches  the  contrary.  Students  are  prone  to  believe  that 
with  one  of  the  elaborate  automatic  microtomes,  good  sections  may 
be  made  with  any  kind  of  an  edge  on  the  knife.  It  is  forgotten  that 
the  knife  is  the  most  important  part;  all  the  other  mechanism  is  simply 
its  servant. 

For  sharpening,  select  a  fine  yellow  Belgian  hone,  and  a  very  fine 
Arkansas  hone.  As  a  rule  hones  from  the  factory  are  not  sufficiently 


CH.  XI]  MICROTOMES  AND   SECTION   KNIVES  373 

plane.  They  may  be  flattened  by  rubbing  them  on  a  piece  of  plate 
glass  covered  with  moderately  fine  emery  or  carborundum  wet  with 
water.  Round  the  corners  and  edges  of  the  hones  on  the  plate  glass 
or  on  a  grindstone.  In  using  the  Belgian  hone  for  sharpening  knives, 
wet  the  surface  well  with  a  moderately  thick  solution  of  soap.  With 
the  Arkansas  stone  use  some  thin  oil  —  xylene  or  kerosene  mixed 
with  a  little  olive  oil  or  machine  oil. 

Honing.  Before  honing  a  section  knife,  make  sure  that  the  edge 
is  smooth;  that  is,  that  it  is  free  from  nicks.  Test  this  by  shaving 
off  the  surface  of  a  block  of  paraffin.  If  nicks  are  present  the  cut 
surface  will  show  scratches.  It  is  advisable  also  to  look  at  the  edge 
of  the  knife  with  a  magnifier  and  with  a  low  power  (50  mm.)  objective. 
If  nicks  are  present  remove  them  by  drawing  the  edge  along  a  very 
fine  Arkansas  hone. 

A  saw  edge  may  be  all  right  for  rough  cutting  and  for  shaving 
razors,  but  if  one  wishes  to  get  perfect  sections  IJJL  to  loju,  in  thick- 
ness a  saw  edge  will  not  do.  In  removing  the  nicks  one  should 
of  course  bear  on  very  lightly.  The  weight  of  the  knife  is  usually 
enough. 

In  honing  use  both  hands;  draw  the  knife,  edge  foremost,  along  the 
hone  with  a  broad  curved  motion.  In  turning  the  knife  for  the  re- 
turn stroke,  turn  the  edge  up,  not  down.  Continue  the  honing  until 
the  hairs  on  the  arm,  wrist,  or  hand  can  be  cut  easily  or  until  a  hair 
from  the  head  can  be  cut  within  5  mm.  from  the  point  where  it  is 
held.  The  sharper  the  knife  becomes  the  lighter  must  one  bear  on. 
One  should  also  use  the  finest  stone  for  finishing.  If  one  bears 
on  too  hard  toward  the  end  of  sharpening,  the  edge  will  be  filled  with 
nicks. 

In  honing  and  stropping  large  section  knives,  there  has  come  into 
use  during  the  last  few  years  the  so-called  " honing  backs."  These 
elevate  the  razor  slightly,  so  that  the  wedge  is  blunter  and  one  does 
not  have  to  grind  away  so  much  steel. 

Strop.  A  good  strop  may  be  made  from  a  piece  of  leather  (horse- 
hide)  about  50  cm.  long  and  5  to  6  cm.  wide,  fastened  to  a  board  of 
about  the  same  size. 

The  strop  is  prepared  for  use  by  rubbing  into  the  smooth  surface 


374  MICROSCOPIC  SECTIONING  [Cn.  XI 

some  carborundum  powder,  i.e.,  6o-minute  carborundum,  that  which 
is  so  fine  that  it  remains  in  suspension  in  water  for  60  minutes,  or  one 
may  use  diamantine  or  jewelers'  rouge. 

Stropping.  With  the  back  foremost  draw  the  knife  lengthwise 
of  the  strop  with  a  broad  sweep.  For  the  return  stroke  turn  the 
edge  up  as  in  honing.  Continue  the  stropping  until  a  hair  can  be 
cut  i  to  2  centimeters  from  where  it  is  held.  (See  also  the  hones  and 
strops  and  the  methods  of  procedure  recommended  in  the  catalogues 
of  microscopical  manufacturers.) 

§  607.  Free-hand  sectioning.  —  To  do  this  one  grasps  the  section 
knife  in  the  right  hand  and  the  object  in  the  left.  Let  the  end  to  be 
cut  project  up  between  the  thumb  and  index  finger.  One  can  let 
the  knife  rest  on  the  thumb  or  index  finger  nail  and  with  a  drawing 
cut  make  the  section  across  the  end  of  the  piece  of  tissue.  By  prac- 
tice one  learns  to  make  excellent  sections  this  way.  If  the  whole 
section  is  not  sufficiently  thin,  very  often  a  part  will  be  and  one  can 
get  the  information  needed. 

§  608.  Sectioning  with  a  hand  or  table  microtome.  —  The  tissue  is 
held  by  the  microtome  and  moved  up  by  means  of  a  screw.  The 
knife  rests  on  the  top  of  the  microtome  and  is  moved  across  the  tissue 
by  the  hand.  Microtomes  of  this  kind  are  excellent.  No  one  need 
wait  for  expensive  automatic  microtomes  to  do  good  sectioning. 
With  a  good  table  microtome,  the  knife  being  guided  by  the  hand 
or  hands  of  the  operator,  he  can  make  straight  cuts  as  for  paraffin 
sectioning,  or  drawing  cuts  as  for  collodion  work. 

§  609.  Sectioning  with  a  freezing  microtome.  —  In  this  method  of 
sectioning  the  tissue  is  rendered  firm  by  freezing  and  the  sections  are 
cut  rapidly  by  a  planing  motion  as  with  paraffin.  Now  the  most 
usual  freezing  microtome  is  one  in  which  the  freezing  is  done  with 
escaping  liquid  carbon  dioxid.  The  knife  should  be  very  rigid.  A 
carpenter's  plane  blade  is  often  made  use  of.  The  tissue  may  be 
either  fresh  or  fixed.  If  alcohol  has  been  used  it  must  be  soaked  out 
of  the  tissue  by  placing  it  in  water.  Sometimes  tissues  are  infiltrated 
a  day  or  two  in  thick  gum  arabic  mucilage  before  freezing.  Drop  a 
little  thick  mucilage  on  the  top  of  the  freezer,  put  the  tissue  in  the 
mucilage,  and  turn  on  a  small  amount  of  carbon  dioxid.  It  will 


CH.  XI]        PREPARATIONS    BY   THE   PARAFFIN   METHOD  375 

soon  freeze  the  mucilage  and  the  tissue,  as  shown  by  the  white  appear- 
ance. When  frozen,  cut  the  tissue  rapidly.  It  is  well  to  have  an 
assistant  turn  the  feed  screw  up  while  the  sections  are  cut.  When 
20  or  30  sections  are  cut  place  them  in  water  or  normal  salt  solution. 
This  is  a  rapid  method  of  getting  sections  much  used  in  pathology 
where  quick  diagnoses  are  demanded.  In  normal  histology  the  freez- 
ing microtome  is  used  mostly  for  organs  or  parts  of  greatly  varying 
density.  For  example,  if  one  wishes  sections  of  the  finger  and  finger 
nail,  this  apparatus  offers  about  the  only  means  of  getting  good 
sections.  In  that  case  the  bone  is  decalcified  before  trying  to  make 
the  sections  (§  559). 

Frozen  sections  are  also  very  useful  for  demonstrating  the  presence 
of  fat  by  staining  with  Sudan  III. 

THE  PARAFFIN  METHOD  OF  SECTIONING 

§  610.  Object  of  the  paraffin.  —  In  the  early  periods  in  histology 
great  difficulty  was  encountered  in  making  good  sections  of  organs 
and  parts  of  organs,  because  the  different  tissues  were  very  unlike  in 
density.  At  first  tallow  and  beeswax,  elder  pith,  liver,  and  various 
other  substances  were  used  to  enclose  or  surround  the  object  to  be 
cut.  This  gave  support  on  all  sides,  but  did  not  render  the  object 
homogeneous.  In  the  early  sectioning,  a  great  effort  was  made  to 
keep  all  imbedding  material  from  becoming  entangled  in  the  meshes 
of  the  tissue.  This  was  guarded  against  by  coating  the  object  with 
mucilage,  and  hardening  it  in  alcohol.  This  mucilage  jacket  kept 
the  tissue  free  from  infiltration  by  the  imbedding  mass  and  itself  was 
easily  gotten  rid  of  by  soaking  the  sections  in  water. 

A  great  advance  was  made  when  it  was  found  that  the  imbedding 
mass  could  be  made  to  fill  all  the  spaces  between  the  tissue  elements 
and  surround  every  part,  the  tissue  assuming  a  nearly  homogeneous 
consistency,  and  cutting  almost  like  the  clear  imbedding  mass.  Cocoa 
butter  was  one  of  the  first  substances  to  be  used  for  thus  "infiltrat- 
ing" the  tissues.  The  imbedding  mass  must  usually  be  removed 
before  the  staining  and  mounting  processes;  but  in  staining  for 
glycogen  by  the  iodin  method,  the  stain  is  applied  before  the  paraf- 
fin is  removed  (§  575). 


376 


PREPARATIONS   BY  THE   PARAFFIN   METHOD        [Cn.  XI 


§  611.  Infiltration  of  the  tissue  with  imbedding  mass.  —  The  tissue 
to  be  cut  in  this  way  is  first  fixed  by  one  of  the  fixers  used  for  his- 
tology. Several  good  ones  are  given  in  sections  568,  586,  592,  601. 

(A)  The  tissue  is  then  thoroughly  dehydrated  by  means  of  95  %  and 
absolute  alcohol.  For  most  objects,  especially  embryos  and  other 
colorless  objects,  it  is  best,  during  the  de- 
hydration, first  to  use  dilute  alcoholic  eosin 
(§  56 2), as  the  most  delicate  part  shows  when 
one  cuts  the  sections.  Leave  the  piece  of 
tissue  to  be  cut  overnight  in  alcoholic  eosin, 
and  a  few  hours  in  uncolored  95  %  alcohol, 
using  20  times  as  much  alcohol  as  tissue. 
For  the  final  dehydration  it  should  be  left 
in  absolute  alcohol  four  or  five  hours  or  over- 
night, depending  on  the  size  of  the  object. 

(B)  Remove  the  alcohol  by  a  solvent  of 
the  imbedding  mass;  that  is,  by  some  sub- 
stance which  is  miscible  with  both  alcohol 
and  the  imbedding  mass.  Cedar-wood  oil 
is  most  generally  used,  but  pure  xylene, 
chloroform,  and  carbol-xylene  are  also  used, 
—  the  chloroform  and  carbol-xylene  when 
osmic  acid  fat  is  to  be  retained  in  the  tissue. 
Leave  the  tissue  in  cedar  oil  or  other 
clearer  until  the  tissue  sinks  and  the  thin 
parts  of  the  specimen  become  translucent. 
If  the  tissue  does  not  sink  after  a  time  it 
means  that  the  tissue  was  not  dehydrated. 
Of  course  this  does  not  apply  to  lung  or  other  spongy  tissue  contain- 
ing much  air.  It  is  well  to  change  the  cedar  oil  or  other  clearer 
once.  The  used  cedar  oil  may  be  left  in  an  open  bottle  for  the 
evaporation  of  alcohol  and  used  over  and  over  again. 

(C)  Displace  the  cedar  oil  or  other  clearer  by  melted  paraffin  wax. 
When  the  tissue  is  saturated  with  the  oil  transfer  it  to  an  infiltrating 
dish  (fig.  220)  containing  melted  paraffin.  Place  in  a  paraffin  oven 
(fig.  220)  and  keep  the  paraffin  melted  for  from  two  hours  to  three 


FIG.  219.     KINGSBURY'S 
PARAFFIN  MELTING  OVEN. 

(From  the  Anatomical 
Record). 

1  Upper   part  of    the 
oven  containing  the  covered 
pitcher  for  the  paraffin. 

2  Lower  part  contain- 
ing the  incandescent  lamps 
and  supply  cable  (c).     The 
oven  is  well  insulated  by 
asbestos.      Depending    on 
the    temperature    of    the 
room,  one  or  both  lamps 
can  be  used  to  keep  the 
paraffin  melted. 


CH.  XI]       PREPARATIONS  BY  THE  PARAFFIN  METHOD 


377 


days,  depending  on  the  size  and  character  of  the  piece  to  be  imbedded. 
If  the  tissue  was  thoroughly  dehydrated  and  well  saturated  with  cedar 
oil,  the  melted  paraffin  permeates  the  whole  piece. 

§  612.  Imbedding  in  paraffin  wax.  —  When  the  object  is  thoroughly 
infiltrated  imbed  as  follows:  Make  of  strong  writing  paper  a  box 
considerably  larger  than  the  piece  to  be  imbedded.  Nearly  fill  the 
box  with  paraffin  wax,  place  on  a  copper  heater  (fig.  226),  and  allow 
to  remain  until  bubbles  appear 
in  it.  Put  the  box  on  cold  water 
until  a  thin  stratum  of  paraffin 
solidifies  on  the  bottom.  Take  the 
piece  of  tissue  from  the  infiltrat- 
ing dish  (fig.  220)  and  arrange  in 
the  box  for  making  sections  in  a 
definite  direction.  Add  hot  para- 
ffin, if  necessary,  and  then  place 
the  box  on  cold  water.  The  more 
rapid  the  cooling,  the  more  homo- 
geneous will  be  the  block  con- 
taining the  tissue  to  be  cut.  For 
the  best  imbedding  it  is  well  to 
drop  95%  alcohol  on  the  sur- 
face as  soon  as  a  film  has  formed 
in  cooling.  In  warm  climates 
where  cold  water  is  not  easy  to 
procure  for  cooling  the  blocks, 
one  may  float  the  paper  box  on 
95%  alcohol  arid  with  a  pipette 
(fig.  234)  drop  strong  alcohol  on 


FIG.  220.    ELECTRIC  INFILTRATING  OVEN 
WITH  PROJECTING  SPREADING  PLATE. 

(About  one  eighth  natural  size.  See 
also  fig.  228.) 

A  Upper  part  of  the  oven  with  its 
brass  spreading  plate  projecting  8  cm.  to 
the  left. 

B  Base  or  tray  holding  the  oven,  and 
the  infiltrating  and  paraffin  dishes,  (i,  2, 

3,  4,  5,  6-) 

cr  The  electric  cable  and  the  porcelain 
receptacle  for  the  lamp  bulb. 

The  oven  and  tray  are  lined  with  as- 
bestos, but  there  is  none  under  the  spread- 
ing plate.  The  dimensions  are :  Brass  top, 
38  x  1 8  cm.,  3  mm.  thick. 

Oven,  30  cm.  long,  18  cm.  wide,  12.5  cm. 
high.  Tray,  30  x  18  x  2  cm. 


the  sides  of  the  box  and  on  the  top  of  the  paraffin  as  soon  as  a  sur- 
face film  has  formed. 

It  is  very  desirable  to  mark  on  the  box  the  name  of  the  imbedded 
object  and  to  indicate  which  end  or  face  is  to  be  cut  (see  also  §657). 

§  613.  Fastening  the  block  to  a  holder.  —  Use  one  of  the  block 
holders  or  object  discs  furnished  with  the  microtome,  or  a  short 
stove  bolt  (fig.  222).  Heat  the  larger  end  and  press  the  paraffin 
block  against  the  hot  metal  until  it  melts  the  paraffin  Hold  the  two 
together  while  cold  water  flows  over  them.  When  cold  the  block 
is  firmly  cemented  to  the  holder.  Pains  should  be  taken  to  have  the 
axis  of  the  block  parallel  with  the  long  axis  of  the  holder;  and  one 


.378 


PREPARATIONS   BY  THE   PARAFFIN   METHOD        [Cn.  XI 


should  not  cut  the  block  so  short  that  the  holder  comes  in  contact 
with  the  tissue  when  the  paraffin  and  holder  are  cemented  together. 


c\j 


CM 


CNJ 


C\J 


4       L 
1 


FIG.  221.    DIAGRAM  SHOWING  How  TO  MAKE  A  PAPER  Box  FOR  IMBEDDING. 

j,  /,  /,     Lines  for  the  first  folds;   these  make  three  longitudinal  strips. 
2,  2,  2,     Lines  for  the  second  folds;   these  make  three  transverse  strips, 
j,  3,  3,     Lines  showing- where  the  corner  folds  are  made. 
4,  4,  4,     The  folds  for  the  projecting  end  or  label. 

B  Bottom,  S  Side,  E  Ends  and  L  Label  of  the  box.     The  bottom  occupies  ?f 
of  the  area. 

A  clamp  is  sometimes  used  for  holding  the  paraffin  block. 
§  614.   Trimming  the  end  of  the  block  for  sectioning.  —  Sharpen 
the  end  to  be  cut  in  a  pyramidal  form,  being  sure  to  leave  2  milli- 


FIG.  222.    CLAMP  FOR  STOVE  BOLTS  TO  BE  USED  AS  HOLDERS  FOR  PARAFFIN 

BLOCKS. 


A  Face  and  B  Sectional  view  with  a  stove  bolt  in  position. 


CH.  XI]        PREPARATIONS  BY  THE  PARAFFIN  METHOD 


379 


meters  or  more  of  paraffin  over  the  tissue  at  the  end  as  well  as  on 
the  sides.  The  block  is  trimmed  in  a  pyramidal  form,  so  that  it 
will  be  rigid.  Take  particular  pains  that  the  opposite  faces  at  the 
end  of  the  block  are  parallel  and  all  the  corners  right  angles. 

In  some  laboratories,  Dr.  McClung's  for 
example,  a  cubical  block  of  metal  attached 
to  a  rod  is  placed  in  the  knife  holder  of  the 
microtome  and  the  four  sides  of  the  im- 
bedding mass  trimmed  with  great  exactness 
by  the  use  of  a  straight-edged  scalpel,  or 
better  by  a  small  chisel,  the  cube  of  metal 
serving  as  a  guide.  As  the  metal  cube  can 
be  slid  along  in  the  knife  holder,  and  the 
imbedded  tissue  can  be  raised  and  lowered 
by  turning  the  wheel  of  the  microtome,  im- 
bedding masses  of  large  and  small  sizes  can 
be  trimmed  by  the  same  metal  guide.  This 
guide  for  trimming  is  a  great  help  in  get- 
ting straight  ribbons,  and  consequently 
good  series. 

§  615.  Making  paraffin  sections.  —  Put 
the  paraffin  block  or  the  metal  holder  in 
the  clamp  of  the  microtome.  Arrange  the 
block  so  that  one  side  of  the  pyramidal 
end  is  parallel  with  the  edge  of  the  knife; 
then  tighten  the  clamp;  and  if  an  automatic 
microtome  is  used,  make  sure  that  the  sec- 
tion knife  is  also  tightly  clamped  by  the 
proper  set  screws.  It  is  well  to  have  the  knife  lean  slightly  toward 
the  paraffin  blocks. 

The  knife  edge  meets  the  paraffin  squarely,  as  in  planing.  The 
thickness  of  section  is  provided  for  in  the  automatic  microtome  by 
the  indicator,  which  may  be  set  for  any  desired  thickness,  or  one 
can  turn  up  the  screw  by  hand  in  the  table  microtome.  The  par- 
affin and  its  contained  tissue  are  cut  in  a  thin  shaving.  If  the 
tissue  was  stained  in  toto  with  eosin,  as  suggested  in  §  611  A,  it  is 


i      2      3 

FIG.  223.   SCALPEL  BLADES. 

i,  2  with  curved  edges 
for  cutting  ribbons;  j,  with 
straight  edge  for  trimming 
paraffin  blocks. 


PREPARATIONS   BY  THE   PARAFFIN  METHOD        [Cn.  XI 

marked  out  with  great  clearness  in  the  containing  paraffin  (see  also 
§657). 

As  succeeding  sections  are  cut  they  push  along  the  previous  sec- 
tions, and  if  the  hardness  of  the  paraffin  is  adapted  to  the  temperature 
where  the  sectioning  is  done,  the  edges  of  the  successive  sections  will 
be  soldered  as  they  strike.  This  produces  a  ribbon,  as  it  is  called, 


FIG.  224.    SUPPORT  OF  THE  MICROTOME  KNIFE  so  THAT  THE  MOST  OF  THE 
EDGE  CAN  BE  USED. 

and  if  the  paraffin  block  has  been  properly  trimmed  at  the  end  the 
ribbon  will  be  straight  and  even.  If  the  ribbon  is  curved  sideways 
it  indicates  that  one  side  of  the  block  is  thicker  than  the  other  and  the 
sections  are  slightly  wedge  shaped. 

If  the  paraffin  is  too  hard  for  the  room  temperature  and  for  a  given 
thickness  of  section,  the  sections  will  curl;  if  it  is  too  soft,  the  sections 
will  crumple. 

The  thinner  the  sections  the  harder  should  be  the  paraffin  or  the 
cooler  the  sectioning  room;  and  the  thicker  the  sections  and  the  larger 
the  object  to  be  cut,  the  softer  can  be  the  paraffin  and  the  higher 
the  temperature.  If,  then,  the  sections  do  not  ribbon,  make  thinner 


CH.  XFj        PREPARATIONS  BY  THE  PARAFFIN  METHOD  381 

sections  or  work  in  a  warmer  place.  If  the  sections  crumple,  make 
thicker  sections  or  work  in  a  cooler  room.  Of  course  one  can  reimbed 
in  a  more  suitable  hardness  of  paraffin. 

In  -the  season  when  steam  radiators  are  used  one  can  get  almost 
any  desired  temperature  by  sectioning  nearer  or  farther  from  the 
radiator. 

In  the  winter  it  is  a  good  plan  to  warm  the  microtome  and  section 
knife  before  sectioning.  This  can  .be  very  easily  done  by  putting 
a  cloth  over  the  radiator  and  the  microtome  something  like  a  tent. 

§  616.  Electrification  of 
the    paraffin    ribbons.  — 

o  j  .,  .  , 

Some  days  there  is  such 
an  accumulation  of  static 

electricity  in  cutting  the 

n'KKn        f^of   fU  FlG-  225-     FlNE  FORCEPS  FOR  HANDLING  COVER- 

ribbons   that  they  jump  'GLASSES,  RIBBON  SECTIONS,  ETC. 

toward  anything  brought 

near  them.     This  is  very  annoying  and  liable  to  be  so  destructive  to 

many  of  the  sections  that  serial  work  cannot  be  done  with  safety. 

Many  devices  have  been  tried  to  overcome  this  difficulty,  like  burn- 
ing a  gas  jet  near  the  microtome,  boiling  water  near  the  apparatus, 
etc.,  but  the  safest  way  is  to  wait  for  more  favorable  conditions. 

To  overcome  this  electrification,  Dixon  (Jour.  Roy.  Micr.  Soc., 
1904,  p.  590)  recommends  fastening  a  5-milligram  tube  of  radium 
bromide  on  the  knife  near  where  the  sectioning  is  done.  The  radium 
ionizes  the  air  and  renders  it  a  good  conductor,  and  then  the  static  elec- 
tricity cannot  accumulate.  I  have  not  been  able  to  try  this  method. 

§  617.  Storing  paraffin  ribbons.  —  The  most  convenient  method  for 
caring  for  the  ribbons  as  they  are  cut  is  to  place  them  on  a  tray 
(fig.  206-207)  lined  with  a  sheet  of  white  paper.  It  is  important  to 
write  on  the  paper  full  data,  giving  the  name  of  the  tissue,  the  thicV 
ness  of  the  sections,  the  date,  etc.  It  is  well  also  to  number  the  rib- 
bons and  to  indicate  clearly  the  position  of  the  first  section  or  the 
beginning  of  the  ribbon. 

Ribbons  of  sections  on  a  tray  should  be  covered  by  another  tray 
if  one  wishes  to  carry  them  to  another  room.  The  slightest  gust  of 
air  sends  them  flying. 


382 


PREPARATIONS  BY  THE  PARAFFIN  METHOD        [Cn.  XI 


FIG.  226.  LEVELING 
METAL  TABLE  FOR 
SPREADING  SECTIONS  AND 
FOR  IMBEDDING  IN  PAR- 
AFFIN. 


Ribbons  on  trays  may  be  kept  a  long  time,  three  or  four  years  at 
least,  if  they  are  stored  in  a  cool  place.  The  sections  do  not  flatten 
out  quite  as  well  after  standing  a  long  time  as  they  do  soon  after  they 
are  made. 

§  618.  Paraffin  ribbon  winder.  —  As  most  embryos  and  many  or- 
gans which  are  to  be  cut  entire  make  ribbons  much  longer  than  the 
slide  tray,  it  is  necessary  to  cut  the  ribbons 
into  segments  usually  as  they  are  made.  If 
one  grasps  the  ribbon  with  fine  forceps  and 
carries  it  out  from  the  microtome  it  is  liable 
to  break  from  its  weight  when  it  gets  long. 
The  spread  of  the  arms  prevents  a  very  long 
section  also.  If  one  has  to  stop  in  making  a 
series  there  is  liable  to  be  a  section  too  thin 
or  too  thick  when  one  begins  again,  and  fre- 
quently a  section  is  lost.  To  overcome  this  very  radical  defect 
McClung  and  Hance  have  devised  what  they  call  a  "Paraffin  Ribbon 
Winder."  This  consists  of  a  cylinder  (mailing  box  on  an  axle)  on 
which  the  ribbon  is  wound  as  cut,  just  as  thread  is  wound  on  a  spool. 
(For  figures  and  descriptions,  see  Anatomical  Record,  June  20,  1916, 
pp.  523-526;  Trans.  Amer. 
Micr.  Soc.,  Vol.  XXXII, 
1913,  pp.  297-299.) 

§  619.  Spreading  the 
sections  on  water.  —  Par- 
affin sections  are  almost  in- 
variably slightly  wrinkled 
or  folded  in  cutting.  To 
remove  the  wrinkles  one 
takes  advantage  of  the  ex- 
pansion of  paraffin  when 
it  is  warmed.  The  sec- 
tions may  be  floated  on  warm  water,  when  they  will  straighten  out 
and  become  smooth,  or  the  usual  method  is  to  stretch  them  on  the 
slide  upon  which  they  are  to  be  finally  mounted. 

By  spreading  sections  on  a  wet  slide  a  double  operation  is  per- 


A  B 

FIG.  227.    ALCOHOL  LAMP  IN  A  VERTICAL  AND 
AN  INCLINED  POSITION. 


CH.  XI]       PREPARATIONS  BY  THE  PARAFFIN  METHOD 


383 


formed,  viz. :  the  sections  are  made  smooth  and  they  are  also  fastened 
to  the  slide.  Put  a  minute  drop  of  albumen  fixative  on  the  middle 
of  a  slide  and  with  the  ball  of  one  finger  spread  it  over  the  slide, 
making  a  thin  even  layer.  It  cannot  be  too  thin.  It  is  liable  to 
stain  if  it  is  too  thick. 

With  a  pipette  (fig.  234)  put  several  drops  of  water  on  the  slide 
and  then  place  a  piece  of  ribbon 
6n  the  water ;  or  put  the  sections 
on  the  albumenized  slide  and  add 
the  water  afterward.  Heat  the 
slide  carefully  over  a  spirit  lamp 
or  gas  flame,  being  sure  not  to 
melt  the  paraffin.  As  the  water 
warms  the  paraffin  expands  and 
stretches  the  sections  out  smooth. 
A  copper  heating  plate  is  good 
(fig.  226),  but  an  electric  spreader 
is  best  (fig.  22; 


FIGS.  228-229.    ELECTRIC  SPREADING 
PLATE  AND  INFILTRATING  OVEN. 


The  projecting  (About  one  eighth  natural  size.     See  also 

top  enables  one  to  heat  this  oven 

.,!_                     i     i     i  n             Tr  A-     The  upper  part  of  the  oven  with 

with  a  gas  or  alcohol  flame.    If  an  brass  spread^g  g£t  extending  8  cm. 

electric  bulb  is  used,  one  of  30  to  (Plate  38  cm.  long,  18  cm.  wide  and  3  mm. 

40  watts  is  sufficient.    All  desired  th0L)Btii  or  tray  for  mfiitrating  dishes 

temperatures  are  possible  byplac-  (fig.  220). 
ing  the  infiltrating  dishes  nearer 


or  farther  from  the  lamp ;  and  in    %    i    Insulation  where    the  cable  passes 

j.  r  into  the  oven, 

spreading  one  can  pass  from  a      ow    Hole  to  show  when  the  current  is 

point  over  the  lamp  where  the   on,  and  for  ventilation. 

rr  i,  ,v  s    Slide  of  serial  sections  spreading  or 

paraffin  may  melt  to   the  over-   Drying 

hanging  top  which  is  only  just 

warm.  The  dimensions  of  the  oven  giving  optimum  space  and 
the  desired  range  of  temperature  are  about  as  follows:  Length  30  cm.; 
width  1 8  cm.;  height  12.5  cm.  The  brass  spreading  plate  on  top  is 
38  cm.  long,  18  cm.  wide  and  3  mm.  thick.  The  tray  on  the  bottom 
is  about  2  cm.  deep.  The  tray  and  oven  are  lined  with  asbestos. 

§  620.     Drying  the  sections.  —  After  the  sections  are  spread,  drain 
off  most  of  the  water,  arrange  the  sections  with  a  needle  or  scalpel, 


384 


PREPARATIONS  BY  THE  PARAFFIN  METHOD        [Cn.  XI 


and  place  the  slide  in  one  of  the  trays  (figs.  206-207).  Allow  it  to  re- 
main overnight  or  preferably  longer.  The  longer  the  drying  in  air 
the  more  surely  do  the  sections  adhere  to  the  glass  slide;  or  use  the 
drying  oven  (fig.  244). 

If  one  is  in  haste  to  take  the  succeeding  steps  in  the  preparation, 
the  slide  may  be  dried  by  putting  it  into  a  drying  oven  at  38°  to  40°  C. 
for  half  an  hour  or  more. 

Some  tissues  are  very  difficult  to  get  perfectly  smooth,  as  just  dej 
scribed.  If  fine  wrinkles  persist,  one  can  sometimes  overcome  the 
difficulty  by  letting  the  slide  cool  and  then  covering  with  a  piece  of 


FIG.  230.    Two  MICROSCOPES  AND  A  CHALET  LAMP  ON  A  LABORATORY  TABLE. 

(See  also  Fig.  125.) 

(About  one  ninth  natural  size). 

fine  tissue  paper  slightly  moistened;  press  down  firmly  with  the  ball 
of  the  finger  on  the  sections.  Then  take  hold  of  the  edge  of  the 
paper  and  roll  it  off  the  sections. 

As  the  water  dries  out  the  spread  sections  come  in  very  close  con- 
tact with  the  glass  and  adhere  quite  firmly  to  it.  The  thinner  the 
sections  the  more  tightly  do  they  stick. 

§  621.  Deparaffining  in  xylene.  —  This  is  accomplished  by  using  a 
solvent  of  paraffin.  The  best  and  safest  one  to  use  in  a  laboratory 


CH.  XI]        PREPARATIONS  BY  THE  PARAFFIN  METHOD 


38S 


is  xylene.  Benzine,  gasoline,  and  even  kerosene  are  used,  but  xylene 
is  a  powerful  solvent  of  paraffin,  does  not  injure  the  tissue,  and  is 
not  very  inflammable,  due  to  the  large  amount  of  carbon  in  its 


Slides 


Fig.  231.  Fig-  232. 

FIG.  231.    SLIDE  BASKET  OR  RACK  FOR  HANDLING  SERIAL  SECTIONS. 

FIG.  232.    GLASS  STOPPERED  SPECIMEN  JAR  WITH  A  SLIDE  BASKET  OR  RACK 
WITH  A  SPECIMEN  IN  PLACE. 

molecule  (see  §  552)  and  the  consequent  high  boiling  point,  136°  C. 


It  requires  only  a  few  minutes  to  dissolve  paraffin  from  the  sec- 
tions, but  a  day  or  more  in  the  xylene  does  no  harm. 


386 


PREPARATIONS  BY  THE  PARAFFIN  METHOD       [Cn.  XI 


When  the  paraffin  is  removed  the  staining  and  -other  operations 
necessary  for  a  completed  preparation  may  be  undertaken  (see  for 
these  §  639). 

§  622.  Collodionizing  the  sections.  —  Except  for  carmine  stains  and 
perhaps  some  others,  collodion  remains  practically  colorless.  While 
the  sections  remain  quite  firmly  attached  to  the  slide  after  they  have 
been  spread  and  dried,  thick  sections  are  liable  to  come  off  in  the  many 
processes  of  staining,  and  if  one  has  many  sections  on  a  slide  some 
of  them  may  become  loosened.  To  avoid  this,  the  sections  are  covered 
with  a  delicate  layer  of  collodion,  which  holds  them  down  to  the  slide. 
The  early  method  was  to  use  a  soft  brush  and  paint  a  thin  film  over 
the  dried  sections  before  they  were  deparaffined.  Now  the  sections 
are  deparaffined,  and  then,  after  draining  the  xylene  from  the  slide, 
10-15  seconds,  it  is  put  into  a  bottle  containing  f  %  collodion  (§  556). 
In  a  minute  or  more  the  collodion  displaces  the  xylene  and  penetrates 
the  sections  and  forms  a  delicate  veil  over  their  free  surface.  No  harm 
is  done  by  leaving  the  sections  in  the  collodion  a  considerable  time, 
but  a  minute  or  two  is  sufficient.  The  slide  is  removed,  allowed  to 
drain  for  half  a  minute,  and  then  put  into  a  jar  of  67  %  alcohol 
(fig.  232).  The  alcohol  fixes  the  collodion  and  removes  the  ether. 
As  the  67  %  alcohol  does  not  hurt  the  tissue,  it  may  stay  in  the  jar  a 
day  or  more,  if  desired,  but  half  an  hour  suffices. 

The  sections  are  now  ready  for  the  subsequent  staining  and  other 
operations  to  make  a  finished  slide.  One  has  to  remember  that  if 
mucicarmine  (§  549)  is  to  be  used  in  staining,  the  preparation  must 
not  be  collodionized,  as  carmine  stains  collodion. 

§623.    Steps  in  order  for  the  paraffin  method.  - 
Name  No. 


Animal 

Date 

Fixer 

Time  of  fix 

Washed  in  water 

67  %  ale 82  %  ale , 

Decalc.  §  398 67,  82  %  ale 

In  toto  stain 

Washed  in 

67  %  ale 82  %  ale 

95  %  ale.  and  eosin 


Absl.  ale Cedar  oil 

Infilt 

Temp,  bath Imbed,  in. 

Sections  cut j*'s. . .  . 

Temp,  room 

Stains.  . 


Mtd.  in. . 
Remarks. 


CH.  XI]       PREPARATIONS   BY  THE   COLLODION   METHOD  387 

THE  COLLODION  OR  PARLODION  METHOD  OF  SECTIONING 

§  624.  Collodion  method.  —  In  this  method  the  tissue  is  thor- 
oughly permeated  with  a  solution  of  collodion,  which  is  afterward 
hardened.  Unlike  the  paraffin  of  the  paraffin  method,  the  collodion 
(§  555a)  is  not  subsequently  removed  from  the  tissue,  but  always 
stays  in  the  sections.  It  is  transparent  and  does  no  harm. 

The  fixing  and  dehydration  with  95  %  alcohol  is  the  same  as  for  the 
paraffin  method. 

The  paraffin  method  gives  thinner  sections  than  the  collodion 
method  and  for  series  and  large  numbers  of  sections  is  superior. 

The  collodion  method  requires  no  heat  for  infiltration,  and  it  does 
not  render  the  firmer  forms  of  .connective  tissue  so  hard  and  difficult 
to  cut.  It  is  especially  adapted  for  making  sections  of  large  pieces 
of  tissue  or  organs  and  when  thick  sections  are  desired.  It  is  not 
easy  to  cut  sections  less  than  ioju,  with  collodion,  while  with  paraffin 
it  is  possible  to  make  good  ribbons  of  small  objects  of  delicate  texture 
2  JJL  to  3  fj,  in  thickness.  With  a  very  sharp  knife  and  small  delicate 
object,  and  one  of  the  better  forms  of  microtomes,  one  can  cut  short 
paraffin  series  in  i  ju  sections  and  get  perfect  ribbons. 

In  plant  histology  paraffin  is  used  for  cytologic  work,  and  by  many 
whenever  possible.  Collodion  must  be  used  for  the  hard  tissues  and 
is  used  by  preference  in  some  laboratories.  (See  references  in  the 
collateral  reading  at  the  end.) 

Collodion  sectioning  is  sometimes  denominated  the  wet  method,  as 
the  tissue  and  sections  must  always  be  wet  with  some  liquid,  while 
the  paraffin  method  is  called  the  dry  method,  as  the  tissue  once  in- 
filtrated with  paraffin  keeps  in  the  air  indefinitely  and  in  cutting 
the  sections  no  liquid  is  used. 

§  625.  Infiltration  with  ether  alcohol.  —  Transfer  the  piece  of  tissue 
to  be  cut  from  95  %  alcohol  to  a  mixture  of  equal  parts  of  sulphuric 
ether  and  95  %  or  absolute  alcohol,  and  leave  in  this  for  a  few  hours 
or  a  day  or  more,  as  is  most  convenient.  This  is  to  soak  the  tissue 
full  of  a  solvent  of  the  collodion. 

§  626.  Infiltration  with  1|  %  collodion.  —  Pour  off  the  ether 
alcohol  from  the  tissue  and  add  ij%  collodion.  Leave  in  this  over- 
night or  longer  if  the  piece  of  tissue  is  large. 


388  PREPARATIONS  BY  THE  COLLODION  METHOD      [Cn.  XI 

§  627.  Infiltration  with  3  %  collodion.  —  Pour  off  the  i  J  %  collo- 
dion and  put  in  its  place  3%  collodion.  Leave  the  tissue  in  this 
half  a  day  or  longer. 

§  628.  Infiltration  with  6  %  collodion.  —  Pour  off  the  3  %  and  add 
6%  collodion  to  the  piece  of  tissue.  For  complete  infiltration  with 
this  thick  collodion  leave  the  tissue  in  it  for  one  day  at  least.  If 
the  object  is  large  it  is  advantageous  to  leave  it  in  for  a  week  or  two. 

§  629.  Infiltration  in  strong  collodion.  —  Many  workers  recommend 
as  thick  a  solution  as  can  be  made  for  the  final  infiltration,  and  a  long 
stay  (2-3  weeks)  in  the  infiltrating  liquid. 

Many  also  recommend  a  great  many  steps  in  the  process,  commenc- 
ing with  i  %  and  gradually  passing  up  through  increasing  strengths 
till  the  thickest  is  reached. 

§  630.  Imbedding  on  a  cork  or  block.  —  For  imbedding  small  pieces 
use  a  piece  of  wood  (deck  plug),  vitrified  fiber,  glass,  or  a  good  cork 
for  a  holder  and  cover  the  end  with  6  %  collodion  and  let  it  get  well 
set  in  the  air;  then  put  the  piece  of  tissue  on  the  holder  and  drop  8% 
collodion  upon  it  at  intervals  until  it  is  well  covered  all  around. 
If  one  takes  considerable  time  for  this  the  collodion  thickens  greatly 
in  the  air.  This  is  an  advantage,  for  it  gives  a  denser  block  for  sec- 
tioning. After  the  collodion  is  pretty  well  set,  place  holder  and  tissue 
in  a  vessel  with  chloroform  to  harden.  One  can  put  the  preparation 
into  the  chloroform,  or,  if  the  vessel  is  tight,  it  may  be  above  the 
chloroform,  the  vapor  then  acting  as  the  hardener. 

§  631.  Imbedding  in  a  paper  box.  —  If  the  object  is  of  considerable 
size  it  is  best  to  use  a  paper  box  for  imbedding,  as  with  paraffin.  If 
a  very  small  amount  of  vaseline  is  rubbed  on  the  inside  of  the  box,  it 
prevents  the  collodion  from  sticking  to  the  paper  (fig.  221,  §657). 

Put  first  some  of  the  thick  collodion  in  the  box  and  let  it  remain 
in  the  air  until  nearly  solid,  2  to  3  minutes.  Then  arrange  the  speci- 
men to  be  cut  as  for  imbedding  in  paraffin,  and  gradually  add  thick 
collodion  until  the  object  is  well  covered.  Let  the  box  stand  for  a  few 
minutes  in  air;  then  place  it  in  a  dish  like  a  Stender  dish  and  pour  some 
chloroform  on  the  bottom  of  the  dish.  Cover  and  the  collodion  will 
harden,  partly  by  the  chloroform  vapor  and  partly  by  that  which 
soaks  through  the  paper.  It  is  well  to  change  the  chloroform  at 


CH.  XI]       PREPARATIONS  BY  THE  COLLODION  METHOD 


389 


least  once.  The  used  chloroform  will  contain  some  ether  alcohol, 
but  is  good  for  killing  animals. 

After  24  or  48  hours  the  collodion  should  be  firm  all  through.  Then 
it  is  placed  in  67  %  alcohol  where  it  may  be  left  a  day  or  more.  If  it  is 
to  be  left  an  indefinite  time  the  67  %  alcohol  should  be  changed  for  82  %. 

§  632.  Sectioning  by  the  collodion  method.  —  For  this  one  can  use 
a  table  microtome  or  one  of  the  sliding  microtomes.  The  sections 
are  made  with  a  knife  set  obliquely  and  hence  with  a  drawing  cut. 


FIG.  233. 


PERFORATED  SECTION  LIFTER  FOR  HANDLING  SINGLE  COLLODION  OR 
FROZEN  SECTIONS. 


The  holder  with  the  small  piece  of  tissue  is  clamped  in  the  micro- 
tome and  arranged  as  desired;  then  the  sections  are  made  with  an 
oblique  knife  which  is  kept  wet  with  82%  alcohol.  The  best  way 
to  keep  the  knife  wet  is  to  have  a  dropping  bottle  over  the  object, 
the  drops  falling  about  every  two  seconds.  As  the  sections  are  cut 
they  are  drawn  up  towards  the  back  of*  the  section  knife  with  a  soft 
brush.  They  can  be  kept  in  order  in  this  way  and  not  interfere  with 
succeeding  sections. 

Some  operators  in  drawing  the  knife  across  the  tissue  use  a  slight 
sawing  motion.  However  one  proceeds,  the  knife  is  drawn  rather 
slowly,  not  rapidly  as  with  paraffin  work. 

If  the  imbedding  was  done  in  a  paper  box,  remove  the  box  and  trim 
the  collodion  block  suitably.  Dry  the  end  away  from  the  tissue,  wet 
it  with  3  %  collodion.  Use  a  piece  of  wood,  a  cork,  or  other  holder  of 
suitable  size.  Put  some  6%  collodion  on  the  holder  and  let  it  dry 
for  a  minute  or  so;  then  press  the  collodion  block  down  on  the  holder. 
Leave  in  the  air  for  a  minute  or  two  and  then  put  into  67  %  alcohol 


3QO  PREPARATIONS   BY  THE   COLLODION  METHOD       [Cn.  XI 

to  harden  the  cementing  collodion.  After  15  minutes,  or  longer  if 
convenient,  put  the  mounted  specimen  into  the  clamp  of  the  micro- 
tome and  cut  as  above. 

Sometimes  when  the  imbedded  object  is  of  sufficient  size  and  the 
collodion  block  is  firm,  the  block  itself  is  put  into  the  microtome 
clamp,  no  wood  or  cork  holder  being  used. 

§  633.  Transferring  sections  from  the  knife  to  the  slide.  —  When 
one  has  cut  the  number  of  sections  for  one  slide,  they  should  be  trans- 
ferred to  the  slide  as  follows:  Take  a  piece  of  white  tissue  paper 
about  3X6  centirneters  in  size  and  lay  it  on  the  knife  over  the  sec- 
tions. Press  down  slightly  so  the  paper  is  in  contact  with  all  the 
sections.  Take  hold  of  the  paper  beyond  the  edge  of  the  knife  and 
gradually  pull  it  down  off  the  knife. 

If  there  is  the  right  amount  of  alcohol  on  the  knife,  the  sections 
adhere  to  the  paper  and  move  with  it.  This  transfers  the  sections 
from  the  knife  to  a  piece  of  tissue  paper.  Place  the  tissue  paper  with 
the  sections  down  on  the  middle  of  an  albumenized  slide.  Cover 
with  another  piece  of  paper  and  press  down  gently.  This  presses  the 
Sections  against  the  slide  and  absorbs  a  part  of  the  alcohol.  Take 
hold  of  one  edge  of  the  paper  and  lift  it  with  a  rolling  motion  from  the 
slide.  The  sections  should  stay  on  the  slide  (§ 


§  633a.  —  Various  forms  of  paper  have  been  used  to  handle  the  collodion 
sections.  It  should  be  moderately  strong,  fine-meshed,  not  liable  to  shed 
lint,  and  fairly  absorbent.  One  of  the  first  and  most  successful  papers  recom- 
mended is  "  closet  or  toilet  paper/'  Cigarette  paper  is  also  excellent.  In  my 
own  work  the  heavy  white  tissue  paper  has  been  found  almost  perfect  for  the 
purpose.  Ordinary  lens  paper  or  thin  blotting  paper  for  absorbing  the  alcohol 
or  oil  may  be  used  with  it. 

§  634.  Fastening  the  sections  to  the  slide.  —  With  a  pipette,  drop 
95  %  alcohol  on  the  slide  of  sections,  then  use  a  pipette  full  of  abso- 
lute alcohol  if  it  is  at  hand.  Drain  most  of  the  alcohol  away  and 
add  a  few  drops  of  ether  alcohol.  The  collodion  should  melt  and 
settle  down  closely  on  the  slide.  If  the  collodion  does  not  melt  the 
dehydration  was  not  sufficient  and  more  alcohol  must  be  used.  After 
the  collodion  has  melted  down  upon  the  slide  let  the  slide  remain  a 
minute  or  two  in  the  air,  and  then  transfer  the  slide  to  a  jar  of  67% 
alcohol  (fig.  232). 


CH.  XI]         PREPARATIONS  BY  THE  COLLODION  METHOD          391 

After  half  an  hour  or  longer  the  preparation  is  ready  to  stain. 

§  635.  The  castor-xylene  method  of  sectioning.  —  The  prepara- 
tion of  the  tissue  is  the  same  as  described  in  §  625-629,  except  that 
when  the  collodion  is  hardened  in  chloroform  it  is  transferred,  not  to 
alcohol,  but  the  block  is  placed  in  castor-xylene  (§  554).  In  a  few 
days  the  collodion  gets  as  transparent  as  glass  and  one  can  see  the 
tissue  within  with  great  clearness.  It  can  remain  in  the  castor- 
xylene  indefinitely. 

In  cutting  one  proceeds  exactly  as  in  §  632,  except  that  the  block 
is  kept  wet  with  castor-xylene  and  not  with  alcohol.  The  sections 
are  arranged  on  the  knife  and  transferred  to  the  slide  in  the  same  way 
'as  for  alcohol  sectioning  (§  633-634). 

For  fastening  the  sections  to  the  slide,  as  no  water  is  present,  one 
can  add  the  ether  alcohol  at  once.  It  is  advantageous  here  to  have 
a  mixture  of  ether  two  parts  and  absolute  alcohol  one  part  for  melt- 
ing the  collodion  in  these  oil  sections. 

Allow  the  slide  to  remain  in  the  air  till  the  collodion  begins  to  look 
dull ;  then  the  slide  may  be  transferred  to  a  jar  of  xylene  to  remove 
the  oil.  From  the  xylene  it  is  transferred  to  95  %  alcohol  and  then 
the  slide  is  ready  to  be  stained,  etc.,  as  described  below  (§  638). 

§  636.   Steps  in  order  for  the  collodion  method.— 


Name 


No. 


Animal 

95  %  &lc 

Date 

Ether-ale 

Fixer  
Time  of  fix  
Washed  in  water  . 



i£%  col  
6%  col  
Imbedded 

.  -.3%  col  ;  .. 
..8%  col  

67  %  ale  
Decalc.  §  398  .... 

.  .82%  ale  

Chloroform  
Or  castor-xylene  .  . 

.  .  67  %  ale  

67%  ale  
In  tolo  stain  

.  .82%  ale  

Sections  cut  
Stains  

••••*'s  

Washed  in  

Mounted  in  

67%  ale... 

.  .82%  ale..  . 

Remarks.  . 

DOUBLE  IMBEDDING  IN  COLLODION  AND  PARAFFIN 

§  637.  Need  of  double  imbedding.  —  Some  objects  like  ova  with 
considerable  yolk  and  other  objects  in  which  the  different  parts  are 
of  unequal  density  or  very  loosely  bound  together  are  advantageously 


392  STAINING  MICROSCOPIC  OBJECTS  [Cn.  XI 

imbedded  first  in  collodion  so  that  there  will  be  a  tough  matrix  to 
hold  the  parts  in  place,  and  then  for  ease  and  rapidity  of  sectioning 
paraffin  imbedding  is  added. 
Steps  in  double  imbedding: 

1.  Fix  in  any  desired  way. 

2.  Dehydrate  with  absolute  alcohol  half  a  day  or  more. 

3.  Put  into  ether  alcohol  hah"  a  day  or  more. 

4.  Put  into  f  %  collodion  half  a  day  or  more. 

5.  Put  into  2\%  collodion  i  to  2  days. 

6.  Put  into  5  %  collodion  for  one  day  or  longer. 

7.  Imbed  in  the  5%  collodion,  using  a  paper  box  (fig.  221).     Take 
the  precaution  to  lightly  vaseline  the  inside  of  the  paper  box  (§631, 

657). 

8.  Float  the  imbedded  tissue  on  chloroform  in  a  glass  dish. 

9.  When  the  collodion  is  hardened  by  the  chloroform,  remove  the 
paper  box  and  transfer  to  the  castor-xylene  (§  554)  clarifier  to  finish 
hardening  and  clarifying  the  collodion  mass. 

10.  Put  into  melted  paraffin  for  infiltration.     Leave  in  the  infil- 
trating oven  (fig.   220)  a  day  or  two.     There  is  some  advantage, 
according  to  some,  in  transferring  to  pure  xylene  or  to  cedar-wood 
oil  for  half  a  day  before  putting  into  the  imbedding  paraffin.     Sec- 
tion in  ribbons  as  with  paraffin  (§615). 

The  sections  are  spread  and  stained  exactly  as  for  the  paraffin 
method,  except  that  carmine  cannot  be  used  without  staining  the 
collodion. 

STAINING  AND  PERMANENT  MOUNTING 

§  638.  Generalities  on  stains.  —  From  the  standpoint  of  the  object 
to  be  stained,  dyes  may  be  divided  into  two  great  groups: 

(i)  (a)  Those  which  select  out  or  differentiate  certain  parts  of 
the  tissue  and  make  them  prominent.  Such  dyes  are  called  then 
differential  or  selective.  If  the  nucleus  is  the  part  selected,  the  dye  is 
frequently  called  a  nuclear  dye. 

(b)  General  or  counter  stains.  These  stain  all  parts  of  the  tissue, 
and  are  usually  contrasting  in  color;  blue  or  purple  and  bright  red 
are  frequent  combinations,  e.g.  hematoxylin  and  eosin.  There  is  an 


CH.  XI] 


STAINING  MICROSCOPIC  OBJECTS 


393 


appearance  of  differentiation  even  with  a  general  stain,  as  the  denser 
portions  of  the  tissue  seem  more  deeply  stained;  that  is,  there  is  more 
substance  and  more  stain  is  taken  up  and  hence  the  color  is  deeper. 

(2)  From  the  standpoint  of  the  solvent  used  in  preparing  the  stains 
they  are  called  (a)  aqueous,  and  (b)  alcoholic. 

If  one  uses  an  aqueous  stain  the  object  must  be  well  wet  with  water 
before  the  stain  is  applied,  and  afterward  well  washed  with  water 
before  put  again  into  alcohol.  If  an  alcoholic  stain 
is  used  the  object  to  be  stained  should  be  from 
alcohol  of  the  same  strength  as  that  used  in  mak- 
ing the  dye.  The  dye  is  also  washed  away  from 
the  tissue  with  the  same  strength  of  alcohol;  it 
may  then  be  put  into  the  stronger  alcohols  for 
dehydration. 

With  reference  to  the  now  much  used  anilin 
dyes,  Wright,  Principles  of  Microscopy,  p.  34, 
gives  this  excellent  general  statement:  " Anilin 
dyes  may  be  regarded  as  salts  containing  a  color- 
ing element  or  chromophor,  united  to  a  base  or 
acid,  according  as  the  chromophor  in  question 
possesses,  in  the  particular  case,  acid  or  basic 
properties.  In  the  case  where  the  chromophor 
functions  as  an  acid,  the  dye  is  denoted  an  acid 
dye  (e.g.  eosin).  In  the  case  where  the  chromophor 
functions  as  a  base,  the  dye  is  designated  a  basic 
dye."  Eosin  is  used  as  an  example  where  the 
chromophor  functions  as  an  acid  and  methylene  blue  where  the 
chromophor  functions  as  a  base. 

The  tissue  elements,  and  their  parts  are  named  from  their  affinity 
for  acid  or  basic  dyes.  For  example,  in  the  blood,  the  red  corpuscles 
and  the  granules  of  some  of  the  leucocytes  have  an  affinity  for  acid 
chromophores  and  hence  stain  strongly  with  eosin.  They  are  accord- 
ingly said  to  be  acidophil  or  oxyphil,  sometimes  also  eosinophil.  The 
nuclei  of  all  the  leucocytes,  and  of  the  red  corpuscles  when  nucleated, 
and  the  granules  of  some  of  the  leucocytes,  have  an  affinity  for  basic 
dyes  and  hence  stain  with  methylene  blue,  and  are  designated  basophil. 


FIG.  234.  REAGENT 
BOTTLE  WITH  PI- 
PETTE. 


394 


STAINING  MICROSCOPIC  OBJECTS 


[CH.  XI 


§  639.  Staining  with  hematoxylin.  —  Take  a  slide  of  sections  pre- 
pared by  the  paraffin  or  the  collodion  method  from  the  jar  of  alcohol 
and  plunge  it  into  a  vessel  of  water  to  remove  the  alcohol.  For  stain- 
ing put  the  slide  of  sections  into  a  jar  or  shell  vial  of  the  hematoxylin 
solution  or  one  can  lay  the  slide  flat  on  the  staining  rack  or  some  other 
support  and  add  the  stain  to  the  sections  (fig.  235-236).  It  usually 
takes  from  2  to  10  minutes  to  stain  sufficiently  with  hematoxylin. 

A  good  plan  when 

.^^S-.^^^^^K^^N  one  is  learning  the 

i^v.:;>.  process  is  to  wash  off 

the  stain  after  one 
minute,  either  with 
a  pipette  or  by  put- 
ting the  slide  in  a 
dish  of  water.  Wipe 
off  the  bottom  of  the 
slide  and  put  it  un- 
der the  microscope. 
Light  well,  use  a  low 
power,  and  one  can 


see  the  nuclei  stained 
a  bluish  or  purple 
color,  as  hematoxy- 
lin is  a  nuclear  dye. 
If  the  color  is  faint, 
continue  the  stain- 
ing until  the  nuclei  stand  out  boldly.  Sometimes  it  takes  a  long 
time  to  stain  well  with  hematoxylin.  In  such  a  case  the  jar  of  stain 
may  be  put  into  the  paraffin  oven  and  the  heat  will  accelerate  the 
staining.  One  may  also  heat  the  individual  slides  as  for  spreading 
sections,  but  one  must  be  careful  not  to  let  the  stain  dry  on  the 
sections.  As  the  stain  evaporates  add  fresh  stain  with  a  pipette. 

When  the  sections  are  well  stained  wibh  hematoxylin,  wash  off 
the  hematoxylin  with  water.  If  the  slide  is  allowed  to  stand  some 
time  in  ordinary  water  the  color  is  likely  to  be  brighter.  This  is 
due  to  the  action  of  the  alkali  (ammonia,  etc.)  usually  present  in  natu- 


FIG.  235. 


BOWL  WITH  DRAINING  RACK  AND  FUNNEL 
FOR  STAINING  SECTIONS. 


CH.  XI] 


STAINING  MICROSCOPIC  OBJECTS 


395 


FIG.  236. 


SMALL  AQUARIUM  JARS  FOR  STAIN- 
ING SERIAL  SECTIONS. 


ral  waters.     One  could  use  distilled  water,  adding  a  few  drops  of  a 
saturated  solution  of  lithium  carbonate. 

Dehydrate  in  95%  alcohol  and  absolute  if  necessary;  clear  and 
mount  in  balsam  as  described  in  §  513. 

Hematoxylin  is  so  nearly  a  pure  nuclear  stain  for  most  tissues  and 
organs  that  the  cell  bodies  are  not  very  evident  with  this  alone;  hence 
some  counterstain  is  gen- 
erally used  also. 

§  640.  Counterstaining 
with  eosin.  —  One  of  the 
solutions  of  eosin  (§  562) 
is  dropped  upon  the  sec- 
tions after  the  hematoxy- 
lin  has  been  washed  away 

with   water.     This    stains 

R     Rack  for  the  top  of  the  jar  and  contain- 
almost      instantly.      One      ing  a  small  draining  funnel. 

rarely  needs  to  stain  with         At  the  left  is  a  sPirit  lamP  used  as  a  balsam 

bottle. 

eosin  over    10  or  30   sec- 
onds.    The  excess  stain  is  then  washed  away  with  pipette  or  by  dip- 
ping the  slide  into  water. 

§  641.  Dehydrating  and  clearing.  —  Put  the  slide  directly  into 
95%  alcohol  after  it  is  rinsed  with  water.  Leave  it  in  the  alcohol 
a  short  time  and  transfer  to  fresh  95  %  alcohol  or  to  absolute  alcohol 
a  few  seconds,  10-20.  One  must  not  leave  the  sections  too  long  in 
the  alcohol  or  the  eosin  will  dissolve  out. 

Remove  the  slide  from  the  alcohol  and  put  it  into  a  jar  of  clearer 
(§  552)  °r  Put  it  on  the  rack  (fig.  235-236)  and  add  enough  clearer  to 
cover  the  sections.  Soon  the  clearer  will  displace  the  alcohol  and 
make  the  sections  translucent.  It  usually  requires  only  half  a  minute 
or  so.  The  clearer  is  drained  off  and  balsam  put  on  the  sections, 
and  then  a  clean  cover-glass  is  added.  One  soon  learns  to  use  the  right 
amount  of  balsam.  It  is  better  to  use  too  much  than  too  little  (§  513). 

§  641a.  —  In  the  past  the  plan  for  changing  sections  from  95  %  alcohol  to 
water,  for  example,  has  been  to  run  them  down  gradually,  using  75,  50  and 
35%  alcohol,  successively.  Each  percentage  may  vary,  but  the  principle  of 
a  gradual  passing  from  strong  alcohol  to  water  was  advocated.  On  the  other 
hand  I  have  found  that  the  safest  method  is  to  plunge  the  slide  directly  into 


396  STAINING  MICROSCOPIC  OBJECTS  [Cn.  XI 

water  from  the  95%  alcohol.  The  diffusion  currents  are  almost  or  quite 
avoided  in  this  way.  There  is  no  time  for  the  alcohol  and  water  to  mix;  the 
alcohol  is  washed  away  almost  instantly  by  the  flood  of  water.  So  in  de- 
hydrating after  the  use  of  watery  stains,  the  slide  is  plunged  quickly  into  a  jar 
of  95  %  alcohol.  The  diffusion  currents  are  avoided  in  the  same  way,  for  the 
water  is  removed  by  the  flood  of  the  alcohol.  This  plan  has  been  submitted 
to  the  severe  test  of  laboratory  work,  and  has  proved  itself  perfectly  satis- 
factory (1895-1908). 

§  642.  Counterstaining  with  the  eosin  in  the  clearer.  —  With  this 
method  the  eosin  is  dissolved  in  the  carbol-xylene  clearer  (§  552a), 
and  the  hematoxylin  stained  sections  are  dehydrated  with  95  %  alco- 
hol and  absolute  alcohol  if  necessary  and  then  placed  in  the  clearer. 
The  sections  are  cleared  and  stained  in  eosin  at  the  same  time.  It 
usually  takes  half  a  minute  or  more  for  the  double  process.  When 
the  sections  are  clear  and  sufficiently  red,  the  slide  is  removed  and  the 
clearer  drained  off  by  holding  in  the  forceps  or  in  the  draining  funnel 
(fig.  235-236).  Then  the  balsam  is  added,  and  covered  as  described 
above. 

It  is  a  good  plan  to  rinse  off  the  stained  clearer  by  pure  xylene 
before  adding  the  balsam.  This  is  not  absolutely  necessary,  how- 
ever. 

§  643.  Hematoxylin  and  picro-fuchsin.  —  Picro-fuchsin  is  so  selec- 
tive in  its  general  staining  that  it  is  frequently  used  after  hematoxylin. 
The  hematoxylin  staining  should  be  intense  and  after  the  hematoxylin 
is  washed  away  add  the  picro-fuchsin  (§587).  It  takes  only  a  few 
seconds  for  it  to  act,  10  to  30  seconds.  Wash  with  distilled  water, 
or  natural  water  very  faintly  acidulated.  The  acid  fuchsin  is  very 
sensitive  to  alkalies  and  fades  easily. 

Dehydrate  in  95  %  and  absolute  alcohol,  clear  and  mount  in  acid 
balsam.  Acid  balsam  injures  hematoxylin,  but  is  necessary  for  the 
red  in  the  picro-fuchsin. 

Look  out  for  mercuric  chlorid  crystals  in  the  sections  (§  654). 

§  644.  Hematoxylin  and  mucicarmine.  —  Tissues  and  organs  are 
best  fixed  in  Zenker's  or  mercuric  chlorid.  Small  intestine  is  one  of 
the  most  striking  and  instructive  organs  for  this  double  stain.  Make 
the  sections  by  the  paraffin  method,  but  do  not  fasten  them  to  the 
slide  with  collodion,  for  collodion  stains  with  mucicarmine  (§  549) . 

Stain  i  to  24  hours  in  mucicarmine.     Wash  off  the  stain  with  water 


CH.  XI]  STAINING  MICROSCOPIC  OBJECTS  397 

and  then  stain  with  hematoxylin.  Do  not  stain  too  deeply.  Wash 
with  water,  dehydrate,  clear  and  mount  in  natural  balsam.  Nuclei 
will  be  bluish  or  purple  and  the  cells  containing  mucus  will  be  rose  red. 
The  goblet  cells  of  the  villi  stand  out  like  small  red  goblets,  and  if 
any  mucus  is  streaming  out  of  them  it  will  be  red. 

§  645.  Elastic  tissue  stain.  —  Take  a  slide  of  sections  made  either 
by  the  paraffin  or  the  collodion  method  from  alcohol  and  put  the  slide 
into  a  jar  or  a  shell  vial  of  the  stain.  This  is  an  alcoholic  stain,  hence 
the  sections  should  not  be  washed  in  water.  Allow  the  stain  to  act 
from  a  half  hour  to  an  hour.  Wash  off  the  superfluous  stain  with 
95%  alcohol  from  a  pipette  or  by  rinsing  in  a  jar  of  95%  alcohol. 
It  is  better  in  either  case  to  use  the  pipette  and  clean  alcohol  for  the 
final  washing. 

This  stain  alone  gives  a  bluish  tone  to  the  entire  tissue,  the  elastic 
tissue  being  stained  a  very  deep  blue.  For  greater  contrast  and  to 
bring  out  the  white  fibrous  tissue,  muscle,  etc.,  counterstain  with 
picro-fuchsin  of  one-fourth  the  strength  given  in  the  regular  stain 
(i.e.,  picro-fuchsin  i  part,  distilled  water  3  parts). 

Dip  the  slide  of  sections  into  distilled  water,  and  then  into  a  shell- 
vial  of  the  stain.  Stain  15  to  30  seconds  on  the  average.  Wash  in 
distilled  water  and  dehydrate  in  95  %  alcohol  and  absolute  if  necessary, 
then  clear  in  carbol-xylene  and  mount  in  acid  balsam  (§  547).  The 
elastic  tissue  should  be  almost  black;  white  fibrous  tissue,  red;  muscle, 
blood,  and  epithelia  yellow  or  yellowish.  Arteries  are  excellent  for 
this  combination. 

§  646.  Combined  elastic  mucicarmine  and  picro-fuchsin  stain.  — 
For  this,  one  should  take  some  object  that  is  known  to  contain  elastic 
tissue,  mucus,  white  fibrous  tissue,  and  muscle.  (The  non-cartilag- 
inous part  of  the  trachea  is  excellent).  The  organ  should  have 
been  fixed  in  mercuric  chlorid  or  Zenker's  fluid  (§  579,  592),  for  this 
preparation.  The  sections  should  be  made  by  the  paraffin  method 
and  no  collodion  should  be  used  for  fastening  the  sections  to  the  slide 
(§  622),  for  collodion  is  stained  by  mucicarmine. 

(1)  Stain  first  in  the  elastic  stain  i  hour.     Wash  well  with  95% 
alcohol  and  then  with  water. 

(2)  Stain  in  a  shell  vial  or  jar  of  mucicarmine  (§  549)  from  i  to  24 


398  STAINING  MICROSCOPIC  OBJECTS  [Cn.  XI 

hours.  Wash  well  with  water,  but  one  must  be  careful  in  treating 
these  sections,  as  they  have  no  collodion  mantle  to  protect  them. 

(3)  Stain  15  to  30  seconds  with  picro-fuchsin  of  one-fourth 
strength  (§645).  Dehydrate  with  95%  and  if  necessary  absolute 
alcohol.  Clear  in  carbol-xylene  and  mount  in  acid  balsam  (§  547). 
The  elastic  tissue  will  be  black  or  blue  black.  Mucus  will  be  carmine 
or  rose  red;  white  fibrous  tissue  will  be  magenta  red;  muscle,  epithe- 
lium, and  blood  will  be  yellow. 

§  647.  Eosin  methylene  blue.  —  One  of  the  best  objects  for  this 
stain  is  a  hemolymph  gland.  Such  a  gland  is  easily  and  surely  found 
by  a  beginner  if  he  takes  the  heart  and  lungs  of  a  veal.  In  the  fat 
around  the  heart  and  behind  the  pleura  will  be  found  red  bodies  look- 
ing almost  like  blood  clots.  Remove  carefully;  fix  in  Zenker's  fluid 
or  mercuric  chlorid  (§  579,  592).  Section  by  the  paraffin  method, 
make  the  sections  5  ju,  and  IOJLI  thick.  Use  collodion  for  insuring  the 
fixation  to  the  slide  (§  622).  Stain  the  sections  5  minutes  in  alcoholic 
eosin  (§  563).  Wash  off  the  eosin  stain  with  water.  (This  is  an 
exception  to  the  generalization  in  §  638.) 

Stain  in  methylene  blue  (§  580)  one-half  to  5  minutes.  Rinse  well 
in  tap  water.  Dehydrate  with  neutral  95  %  alcohol  and  with  absolute 
alcohol.  Work  rapidly  with  only  one  slide  at  once.  Clear  with  pure 
xylene,  mount  in  neutral  balsam  (§  546).  All  nuclei  should  be  blue, 
and  all  red  blood  corpuscles  bright  eosin  red.  If  one  is  successful 
this  is  a  most  striking  and  instructive  preparation.  Spleen  is  also 
very  instructive. 

Eosin-methylene  blue  staining  is  also  excellent  for  demonstrating 
mucus. 

Do  not  forget  that  mercury  is  liable  to  be  present  in  sections  of 
tissue  fixed  with  any  mercuric  fixer.  Remove  it  with  iodized  alco- 
hol (§  576).  This  should  be  done  before  the  staining.  One  can  tell 
whether  the  tissues  contain  mercury  by  looking  at  the  unstained 
sections.  The  mercury  looks  black  by  transmitted  light,  white  by 
reflected  light.  Seen  by  transmitted  light,  the  substance  is  often  in 
the  form  of  delicate  black  pins. 

§  648.  lodin  stain  for  glycogen.  —  Use  tissue  fixed  in  95  %  or  abso- 
lute alcohol.  Cut  by  the  paraffin  method.  Mount  the  sections  in 


CH.  XI]  SERIAL  MICROSCOPIC  SECTIONS  399 

serial  order.  Do  not  use  water  for  spreading  the  sections,  but  one 
of  the  iodin  stains  for  glycogen  (§575).  The  glycogen  will  be  stained 
at  the  same  time  that  the  sections  are  spread. 

Let  the  sections  dry  thoroughly  after  spreading.  Deparaffin  with 
xylene  and  mount  in  yellow  vaseline  or  use  thin  xylene  balsam,  but 
do  not  put  a  cover-glass  over  the  balsam  preparations. 

The  iodin  stain  remains  in  the  spread  sections  for  ten  years  or  longer. 
One  can  restain  any  time  by  putting  the  slide  with  the  spread,  but 
not  deparaffined  sections,  in  a  shellvial  of  the  iodin  stain.  It  is  pos- 
sible also  to  stain  the  nuclei  with  hematoxylin  in  the  same  way.  If 
this  is  done  the  hematoxylin  should  be  used  first  and  washed  off  with 
water  and  the  iodin  stain  be  used  last,  but  not  washed  off  with  water. 

ADVANTAGES  OF  HISTOLOGICAL  SERIAL  SECTIONS 

§  649.  General  on  series.  —  It  is  coming  to  be  appreciated  more 
and  more  that  in  histology  as  well  as  in  embryology  one  can  only  get 
a  complete  knowledge  of  structure  by  having  the  entire  organ  cut  in 
microscopic  sections  and  each  section  mounted  in  order.  Further- 
more, it  is  necessary  to  have  the  organ  cut  in  three  different  planes. 
In  this  way  one  can  see  every  aspect  of  the  structural  elements  and 
their  arrangement  in  the  organs. 

In  single  sections  one  gets  only  a  partial  view.  For  example,  how 
many  students  have  any  other  idea  of  a  ciliated  ceh1  than  that  it  is 
a  cell  with  triangular  outline  with  a  brush  of  cilia  at  the  board  end. 
Probably  many  would  be  puzzled  if  they  had  a  top  view  of  the  ciliated 
end;  and  the  attached  end  would  be  even  more  puzzling. 

It  may  not  be  possible  for  every  worker  to  make  serial  sections 
of  all  the  organs  in  all  the  three  planes,  but  every  one  who  is  working 
seriously  in  histology  can  make  all  his  preparations  serial;  that  is,  the 
sections  which  are  mounted  can  be  in  serial  order;  then  a  puzzling 
appearance  in  one  section  may  be  perfectly  intelligible  in  one  a  little 
farther  along. 

To  get  the  greatest  benefit  from  serial  as  indeed  also  from  single 
sections,  the  sections  should  be  made  in  a  definite  manner;  that  is, 
they  should  be  exactly  across  the  long  axis  of  an  organ  or  parallel 
with  the  long  axis  (Transections  and  Longi sections). 


400 


SERIAL   MICROSCOPIC   SECTIONS 


[Cn.  XI 


Or  with  such  an  organ  as  the  liver,  the  skin,  etc.,  the  sections  may 
be  parallel  with  the  surface  (Surface  Sections)  or  at  right  angles  to 
the  surface  (Vertical  Sections). 

§  650.  Order  of  serial  sections.  —  Some  plan  must  be  adopted  in 
arranging  the  series  or  only  confusion  will  result.  An  excellent  plan 
is  to  arrange  the  short  pieces  of  ribbons  for  a  given  slide  as  the  words 
on  a  page  are  arranged.  That  is,  section  No.  i  is  at  the  upper  left- 
hand  corner.  The  next  row  of  sections  begins  where  the  first  row 
left  off,  etc.  (fig.  237). 

As  the  paraffin  stretches  considerably  one  must  cut  the  ribbons 
into  pieces  considerably  shorter  than  the  cover-glass  to  be  used. 


Pig 


23 


Ss. 


Pig  6 

SI  23 

Sec  253 

\Qfi  260 


1900 


FIG.  237.    A  SLIDE  OF  SERIAL  SECTIONS  SHOWING  THE  ARRANGEMENT  AND  ORDER 
OF  THE  SECTIONS;    ALSO  THE  LABELING  OF  THE  SLIDE. 

Both  the  paraffin  and  collodion  methods  are  adapted  to  the  prepa- 
ration of  series.  The  paraffin  ribbons  are  easier  to  manage  and 
easier  to  make  than  the  serial  sections  in  collodion. 

By  arranging  the  collodion  sections  as  they  are  cut  on  the  knife 
in  collodion  sectioning  (§632),  one  can  put  them  on  the  slide  in  per- 
fect series  by  the  tissue  paper  method  (§  633). 

If  the  sections  are  large,  as  in  cutting  serial  sections  of  the  central 
nervous  system,  the  series  can  be  kept  in  order  in  a  small  dish  by  put- 
ting a  piece  of  tissue  paper  over  each  section  and  piling  them  up.  If 
the  vessel  is  small  enough  the  papers  and  sections  will  not  shift  and 
get  out  of  order.  Or  one  might  put  a  single  section  in  a  Syracuse 
watch  glass  or  a  Petri  dish.  Then  in  mounting  the  sections  can  be 
taken  in  order. 

§  651.  Numbering  the  serial  slides.  —  For  temporary  numbering 
a  fine  pen  with  Higgins'  or  Weber's  waterproof  carbon  ink  serves 


CH.  Xr]  SERIAL  MICROSCOPIC  SECTIONS  401 

well.  If  the  end  of  the  slide  is  varnished,  one  can  write  on  it  as 
well  as  on  paper.  When  the  ink  is  dry  it  should  be  coated  with  thin 
xylene  balsam  or  with  any  good  varnish  like  valspar  i  part,  xylene 
9  parts.  It  is  also  important  to  write  the  number  of  the  slide  with 
a  writing  diamond.  The  double  marking  is  desirable  because  with 
wet  slides  the  diamond  number  is  hard  to  see,  while  the  ink  marks 
are  clearly  visible.  One  is  not  so  liable  to  wipe  off  the  sections  if  the 
ink  mark  is  present. 

FIXING  AND  STAINING  FOR  SERIES 

§  652.  Fixing.  —  The  two  most  used  fixers  for  embryos  are  Zenker's 
fluid  and  formaldehyde  (§568,  592).  For  those  unskilled  in  micro- 
scopic technic,  or  for  one  who  is  exceedingly  busy,  the  best  results 
are  obtained  by  putting  the  embryos  in  formaldehyde  (10  parts  of 
formalin,  the  formalin  of  the  pharmacy,  and  90  parts  water  answers 
well).  If  there  is  plenty  of  this  the  embryos  are  likely  to  be  well  pre- 
served even  though  they  are  left  in  the  membranes,  and  that  is  far 
the  best  way  for  small  embryos. 

§  653.  Fastening  the  sections  to  the  slide.  —  For  all  serial  work 
it  is  especially  desirable  to  fasten  the  sections  to  the  slide  with  collo- 
dion (§  622).  This  should  always  be  done  unless  some  stain  like  car- 
mine is  to  be  used  on  the  slide  after  the  sections  are  fastened.  With 
thin  sections,  if  one  is  careful  enough,  an  entire  series  can  be  carried 
through  without  losing  a  section,  but  with  thick  sections  (15^  and 
thicker)  some  are  almost  sure  to  separate  from  the  slide  if  not  fas- 
tened by  collodion. 

§  654.  Removal  of  mercuric  chlorid  from  sections.  —  It  should  be 
remembered  that  if  a  fixer  containing  mercuric  chlorid  is  used  the 
sections  are  almost  sure  to  contain  mercury.  By  transmitted  light 
the  mercury  appears  dark.  Often  the  appearance  is  as  if  a  multitude 
of  delicate  black  pins  were  in  the  section.  Sometimes  the  mercury 
is  in  rounded  masses.  This  should  be  removed  by  putting  the  slides 
of  sections  into  alcoholic  iodin  (§  576).  After  half  an  hour  or  an  hour 
wash  off  the  iodized  alcohol  with  pure  95  %  alcohol  and  the  sections 
are  ready  for  staining. 

If  the  embryo  was  stained  in  ioto  and  contains  mercury,  the  sec- 


402  SERIAL  MICROSCOPIC   SECTIONS  [Cm  XI 

tions  should  be  passed  from  the  deparaffining  xylene  to  the  iodized 
alcohol  (§  576).  After  half  an  hour  or  more  the  slides  are  passed 
through  pure  95  %  alcohol,  and  back  to  the  xylene  or  to  carbol- 
xylene.  Then  they,  can  be  mounted  in  balsam. 

§  655.  Staining  for  series.  —  There  is  a  great  advantage  in  point 
of  time  and  safety  in  staining  the  entire  embryo  in  some  good  stain 
like  borax  carmine  (§  548) .  Carmine  is  a  very  permanent  stain  also. 
For  bringing  out  special  structural  details  the  sections  are  stained 
on  the  slide  as  described  in  §  639-640.  The  slide  baskets  are  almost 
a  necessity  for  serial  work  (fig.  231-232),  as  the  slides  are  handled 
individually  only  twice,  (i)  when  they  are  spread  and  dried  and  put 
into  the  baskets,  and  (2)  after  all  the  processes  are  complete  and  the 
sections  are  to  be  mounted  in  balsam. 

The  sections  are  mounted  in  balsam  directly  from  the  deparamning 
xylene.  No  alcohol  is  used  unless  it  is  necessary  to  remove  crystals 
of  mercuric  chlorid  (§576,  654). 

COMPLETE  SERIES  or  EMBRYOS  AND  SMALL  ANIMALS  IN  THE  THREE 
CARDINAL  PLANES, — TRANSECTIONS  ;  SAGITTAL  SECTIONS; 
FRONTAL  SECTIONS 

§  656.  Serial  sections  of  entire  animals.  —  With  improvement  in 
means  for  making  thin  sections  of  objects,  the  long  desired  ability  to 
see  the  entire  organism  in  complete  series  is  now  easily  realized. 
What  was  formerly  determined  with  so  much  difficulty  in  dissecting 
embryos  can  now  be  attained  with  ease  in  a  complete  series.  It  is 
almost  too  easy,  and  with  a  lively  imagination  structural  arrangements 
are  described  and  depicted  which  never  actually  existed  in  the  animals 
or  embryos  themselves.  It  is  so  difficult  for  most  people  to  add  the 
third  dimension  accurately  when  working  with  flat  specimens  that  it 
is  now  appreciated  that  the  older  workers  had  a  great  advantage  in 
dissecting  the  entire  animal  or  embryo  because  they  were  there  deal- 
ing with  an  obviously  three-dimensional  object  and  true  relations  in 
space  were  seen.  There  is  now  a  wholesome  tendency  toward  the 
retention  of  the  advantages  of  dissection  of  entire  forms  with  the 
advantages  of  serial  sections.  Hence  embryos  are  now  dissected 
entire  almost  as  much  as  in  the  old  days,  and  enlarged  models  of  the 


CH.  XI]  SERIAL  MICROSCOPIC  SECTIONS  403 

series  are  made  so  that  the  object  can  be  seen  in  three  dimensions, 
the  models  also  serving  to  make  it  easy  to  follow  out  the  relations 
of  parts  with  the  naked  eye.  But  one  should  not  forget  that  a  model, 
like  a  drawing,  is  after  all  only  the  interpretation  of  the  artist  and  the 
thing  itself  must  be  referred  to  whenever  there  is  to  be  real  ad- 
vancement in  knowledge.  Furthermore,  as  it  is  not  possible  to  both 
dissect  and  serial  section  the  same  object,  and  sometimes  very  few 
are  available,  anatomists  have  decided  on  the  three  planes  which 
give  the  greatest  information,  —  transections  or  cross  sections, 
sagittal  sections,  and  frontal  sections.  With  sections  in  these  three 
spatial  planes  it  is  possible  to  gain  some  just  conception  of  the 
actual  relation  of  parts  and  structures  in  the  object. 

§  657.  Orientation  of  imbedded  objects.  —  In  order  that  sections 
may  be  made  in  any  desired  plane  the  object  must  be  so  arranged 
or  oriented  in  the  imbedding  mass  that  one  can  attach  the  imbedding 
block  to  the  microtome  holder,  and  then  arrange  for  sectioning  in  a 
definite  manner.  With  translucent  or  transparent  collodion  where 
the  position  of  the  object  can  be  seen  after  it  is  imbedded,  this  is  not 
particularly  difficult,  but  with  paraffin,  which  is  nearly  opaque,  one 
cannot  see  distinctly  enough  the  position  of  the  object  to  give  the 
exact  arrangement  necessary  to  make  precise  sectioning  possible. 
The  embryo  or  animal  or  other  object  must  therefore  be  arranged 
in  the  imbedding  box  in  a  very  definite  manner. 

To  overcome  the  difficulties  Dr.  Kingsbury,  ten  to  fifteen  years 
ago,  devised  the  method  of  making  a  diagram  of  the  object  to  show 
its  exact  shape  and  position.  (Anat.  Record,  Vol.  XI,  1916,  p.  294). 
The  method  is  as  follows:  A  natural-size  diagram  of  the  object  is 
made  on  the  inside  of  the  bottom  of  the  imbedding  box  before  any 
paraffin  is  put  into  it.  This  is  most  easily  done  before  the  box  is 
folded,  or  the  folded  box  can  be  unfolded  and  made  flat  again.  For 
making  the  diagram  a  soft  lead  pencil  can  be  used  or  one  of  the  or- 
dinary colored  crayons  or  a  colored  glass  pencil.  In  any  case  enough 
of  the  lead  pencil  or  the  crayon  mark  adheres  to  the  paraffin  to  make 
a  clear  diagram  on  it  of  the  object. 

In  imbedding,  the  object  should  be  arranged  exactly  over  the  dia- 
gram. The  solidified  layer  of  paraffin  formed  before  the  object  is 


404  SERIAL  MICROSCOPIC  SECTIONS  [Cn.  XI 

placed  in  the  box  (§  612)  is  no  hindrance,  as  the  diagram  shows 
through  it  clearly. 

For  embryos  and  small  animals,  of  which  serial  sections  are  to  be 
made,  there  should  always  be  a  photograph  natural  size. 

The  diagram  for  orientation  is  easily  made  from  such  a  photograph 
by  the  use  of  the  drawing  shelf  (fig.  247,  A.D.S.,  §  289,  291).  As 
the  embryo  or  animal  is  always  imbedded  with  the  right  side  down, 
left  side  up,  one  must  l?e  sure  to  have  the  diagram  in  the  same  posi- 
tion. This  is  easily  accomplished,  as  one  can  draw  equally  well  with 
the  photographic  print  whichever  side  is  up.  That  is,  if  the  embryo 
was  photographed  left  side  down,  the  print  should  be  face  down  on 
the  drawing  shelf  to  bring  the  diagram  in  the  imbedding  box  with 
the  left  side  up.  On  the  other  hand,  if  the  photograph  was  made 
with  the  embryo  right  side  down,  then  the  print  should  be  face  up 
when  making  the  diagram  on  the  bottom  of  the  imbedding  box. 

With  the  definite  outline  of  the  embryo  or  animal  on  the  bottom  of 
the  imbedding  mass  one  has  a  good  guide  for  arranging  the  object 
for  sectioning  any  desired  plane. 

§  658.  Thickness  of  serial  sections.  —  The  thickness  of  the  sec- 
tions of  a  series  should  be  known  in  all  cases;  and  for  modeling  it  is 
absolutely  necessary  (§665,  669).  The  thickness  usually  depends 
somewhat  upon  the  size  of  the  object  to  be  made  into  series.  If  the 
object  is  small  the  sections  can  be  thin  without  having  an  unmanage- 
able number  of  slides.  With  larger  objects  the  sections  are  naturally 
made  thicker  to  keep  the  length  of  the  series  within  bounds. 

One  of  the  following  thicknesses  will  be  found  to  meet  nearly  all 
requirements  and  make  modeling  easier  than  as  if  some  odd  number 
of  microns  were  used:  5/4,  loju,  i5/z,  20/1,  25/4,  30/^,40^,  soju,  75^,  loo/x. 
Of  course  every  investigator  decides  for  himself  the  thickness  of  sec- 
tion which  will  serve  his  purposes  best. 

§  659.  Arrangement  of  sections  on  the  slide.  —  (i)  A  satisfactory 
and  widely  adopted  method  is  to  arrange  the  the  sections  like  the 
printed  words  in  a  book.  This  brings  the  first  section  at  the  upper 
left-hand  corner  of  the  series,  and  the  last  section  at  the  lower  right- 
hand  corner  (fig.  239). 

(2)   It  is  a  great  advantage  to  have  the  sections  so  arranged  on 


CH.  XI]  SERIAL  MICROSCOPIC  SECTIONS  405 

the  slide  that  under  the  compound  microscope  the  aspects  will  be 
as  in  the  observer's  body;  then  it  will  be  easy  to  locate  objects  at  the 
right  or  left,  dorsal  or  ventral. 

(3)  Remember  that  in  the  ribbons  the  surfaces  are  somewhat 
unlike  in  appearance.     The  lower  surface,  that  is  the  surface  facing 
the  section  knife,  is  shiny,  while  the  opposite  surface  is  dull.     This 
knowledge  is  important,  for  sometimes  sections  get  turned  over  acci- 
dentally.    It  is  unfortunate  to  have  part  of  the  sections  of  a  series 
wrong  side  up. 

(4)  The  aspect  cut  first  will  face  upward  on  the  slide,  that  is,  if 
the  head  is  cut  first  the  cephalic  aspect  will  face  up;   if  the  left  side 
is  cut  first  the  sinistral  aspect  will  face  up,  and  if  the  dorsal  side,  the 
dorsal  face  will  be  up. 

(5)  The  aspect  of  the  embryo  which  first  meets  the  edge  of  the 
knife  will  be  at  the  beginning  of  the  series.     If  arranged  and  cut  as 
here  directed,  transections  would  have  the  right  side  of  each  section 
toward  the  left  on  the  slide  (fig.  239).     Under  the  compound  micro- 
scope it  would  appear  on  the  right. 

For  sagittal  sections  where  the  caudal  end  meets  the  knife,  the  caudal 
end  of  the  section  would  be  toward  the  left  on  the  slide  (fig.  242). 

For  frontal  sections  (fig.  240)  where  the  right  side  meets  the  knife 
edge  first,  the  right  side  of  each  section  will  be  toward  the  left  end 
of  the  slide. 

§  660.  Mounting.  —  Cut  the  ribbons  into  segments  of  equal  length, 
using  preferably  a  curved  knife  (fig.  223).  Transfer  to  albumenized 
slides  with  fine  forceps  (fig.  225).  Make  parallel  with  the  long  axis 
of  the  slide,  and  put  the  first  section  at  the  upper  left-hand  corner 
(fig.  237). 

In  a  word,  decide  on  some  good  plan  for  mounting  series  and  follow 
the  plan  consistently. 

§  661.  Size  of  slides  and  cover-glasses  for  series.  —  (i)  If  the 
object  is  small,  the  standard  slide  25  x  75  mm.  (fig.  185)  is  good  and 
the  cover-glass  can  be  either  22  or  23  mm.  wide  and  50  or  60  mm. 
long.  The  smaller  sizes  are  to  be  preferred  when  convenient,  for  more 
space  is  left  to  the  label,  and  the  cover-glass  is  not  too  near  the  edge 
as  with  wide  covers. 


4o6 


SERIAL  MICROSCOPIC  SECTIONS 


[CH.  XI 


(2)  If  the  embryo  or  animal  is  of  moderate  size,  that  is,  not  over 
30  to  35  mm.  long,  one  can  use  advantageously  the  intermediate  size 
of  slides   (fig.  186),  that  is,  those  38  x  75  mm.     A  suitable  cover- 
glass  is  35  x  50  or  35  x  60  mm. 

(3)  For  objects  of  considerable  size,  i.e.,  over  35  mm.  in  length, 
if  sagittal  or  frontal  sections  are  to  be  made,  and  if  they  are  to  be 
mounted  crosswise,  the  slide  must  be  of  sufficient  width.     Ordinarily 
the  large  standard,  50  x  75  mm.,  will  answer  (fig.  187).     For  the  large 
slides  the  covers  can  be  48  x  60  or  48  x  65  mm.     For  special  large 
sizes  of  object,  special  slides  can  be  made  of  lantern  slide  covers  or 
old  negative  glass,  etc.,  and  for  cover-glasses  one  can  go  back  to  the 
earlier  workers  and  use  mica. 

Do  not  use  too  thick  cover-glasses  or  high  powers  cannot  be  em- 
ployed in  studying  the  sections  (§  76-81). 

TRANSECTIONS  OR  CROSS  SECTIONS 

§  662.  Transactions  are  those  made  by  dividing  the  body  into  sec- 
tions made  across  the  long  axis  of  the  body.     This  divides  the  embryo 


FIG.  238.    SERIAL  TRANSECTIONS. 

At  the  left  is  the  embryo  in  the  imbedding  mass  and  attached  to  the  microtome 
holder. 

At  the  right  is  a  glass  slide  showing  how  the  sections  are  to  be  mounted. 

Imbedded  embryo     It  is  in  the  proper  position  for  transections. 

In  section  i,  the  word  cephalic  shows  that  the  section  is  cephalic  face  up;  the 
caudal  face  rests  on  the  slide.  In  the  middle  section  the  words  indicate  the  edges 
of  the  section.  Under  the  microscope  the  words  will  be  erect.  Invert  the  book 
and  the  appearance  will  be  the  same  as  under  the  microscope. 

or  animal  into  equal  or  unequal  cephalic  and  caudal  segments.  With 
microscopic  sections  of  course  the  segments  of  the  entire  body  are 
very  unequal,  although  each  section  may  be  of  equal  thickness. 

(i)  Imbed  the  embryo  or  animal  with  the. right  side  down,  taking 
the  precaution  to  have  a  layer  of  partly  solidified  paraffin  at  the 


CH.  XI] 


SERIAL  MICROSCOPIC  SECTIONS 


407 


bottom  of  the  box  (§612);  and  arrange  the  object  exactly  over  the 
orientation  diagram  in  the  bottom  of  the  imbedding  box  (§657). 

(2)  Mount  the  block  of  paraffin  containing  the  embryo  so  that 
the  caudal  end  is  next  the  microtome  holder.     The  head  is  then  cut 
first,  and  the  caudal  surface  of  the  sections  will  rest  upon  the  slide, 
bringing  the  cephalic  face  up  (fig.  238). 

(3)  Place  in  the  microtome  so  that  the  right  side  of  the  embryo 
or  animal  meets  the  edge  of  the  knife. 

(4)  Mount  the  sections  like  the  words  in  a  printed  line.     This  will 
bring  the  first  or  most  cephalic  section  at  the  upper  left-hand  corner. 


Homo 


3 
40 


Homo       3 
SI  40 

Sec       493 


20  fj,     504 


1900 


PIG.  239.    A  SLIDE  or  SERIAL  TRANSECTIONS  SHOWING  THE  ARRANGEMENT  AND 
THE  LABELING  OF  THE  SLIDE. 

The  cephalic  face  will  be  up,  and  the  dorsal  aspect  next  the  upper 
edge  of  the  slide. 

Under  the  compound  microscope  the  rights  and  lefts  will  appear 
as  in  the  observer's  own  body,  as  will  also  the  dorsal  and  ventral 
parts. 

FRONTAL  SECTIONS 

§  663.  Frontal  sections.  —  These  are  sections  made  by  dividing 
the  body  into  equal  or  unequal  dorsal  and  ventral  parts. 

(1)  Imbed  the  animal  or  embryo  with  the  right  side  down  in  the 
imbedding  mass  (§612)  ;   and  arrange  the  object  exactly  over  the 
orientation  diagram  in  the  bottom  of  the  imbedding  box  (§657). 

(2)  Mount  the  block  of  paraffin  containing  the  embryo  so  that 
the  ventral  aspect  of  the  embryo  or  animal  is  next  the  disc  of  the 
microtome  holder  (fig.  240).     The  dorsal  part  is  then  cut  first,  and 
the  ventral  surface  of  the  sections  will  rest  upon  the  slide,  bringing 
the  dorsal  face  up. 


408 


SERIAL  MICROSCOPIC  SECTIONS 


[CH.XI 


(3)  Place  in  the  microtome  so  that  the  right  side  of  the  object 
meets  the  edge  of  the  knife  first. 

(4)  Mount  the  sections  like  the  words  in  a  printed  book.    This 
will  bring  the  first  or  dorsalmost  section  in  the  upper  left-hand  corner 


Vontalj 


FIG.  240.  FRONTAL  SERIAL  SECTIONS  SHOWING  THE  ARRANGEMENT  OF  THE 
EMBRYO  IN  THE  IMBEDDING  MASS,  THE  CONNECTION  WITH  THE  MICROTOME 
HOLDER,  AND  THE  POSITION  OF  THE  SECTIONS  ON  THE  GLASS  SLIDE. 

Microtome  Holder  The  metal  disc  and  stem  for  holding  the  imbedded  embryo 
in  the  microtome  while  sectioning. 

Imbedded  Embryo    The  embryo  in  the  proper  position  for  frontal  sections. 

Frontal  Sections    A  slide  showing  the  proper  arrangement  of  frontal  sections. 

i,  2,  j,  4  Serial  order  in  which  the  sections  are  arranged  like  the  words  in  a 
printed  book. 

In  section  /  the  word  dorsal  indicates  that  the  section  has  its  dorsal  face  upward 
away  from  the  slide  while  the  ventral  face  is  down  in  contact  with  the  slide. 

In  section  3,  the  words  cephalic,  caudal,  dextral,  sinistral  are  wrong  side  up  so 
that  they  will  appear  erect  under  the  compound  microscope. 

of  the  series.     The  dorsal  face  will  be  up,  the  right  side  to  the  left, 
and  the  cephalic  end  toward  the  lower  edge  of  the  slide  (fig.  240-241). 


Pig 

4 

~ieT 


Sea        440 
^Ot      453 


19OO 


FIG.   241.     FRONTAL  SERIAL  SECTIONS  SHOWING  THE  ARRANGEMENT  AND  THE 
NUMBERS  OF  THE  SECTIONS  ON  THIS  SLIDE.    THE  SLIDE  is  PROPERLY  LABELED. 

Under  the  compound  microscope  the  cephalic  end  will  be  away  from  the 
observer  or  in  front,  and  the  rights  and  lefts  will  be  as  in  his  own  body. 
If  the  sections  are  too  long  to  mount  crosswise  they  can  be  cut  apart 
and  mounted  lengthwise  of  the  slide,  the  order  being  like  that  of  the 
words  in  a  line  of  print  as  with  all  serial  sections. 


CH.  XI] 


SERIAL  MICROSCOPIC  SECTIONS 


409 


SAGITTAL  SECTIONS 

§  664.  Sagittal  sections  are  those  made  parallel  with  the  long  axis 
of  the  body  and  from  the  dorsal  to  the  ventral  surface,  thus,  dividing 
the  object  into  equal  or  unequal  right  and  left  (dextral  and  sinistral) 
parts. 

(i)  Imbed  the  animal  or  embryo  with  the  right  side  down  in  the 
imbedding  mass  (§612);  and  arrange  the  object  exactly  over  the 
orientation  diagram  in  the  bottom  of  the  imbedding  box  (§657). 


FIG.  242.  SERIAL  SAGITTAL  SECTIONS  SHOWING  THE  POSITION  OF  THE  EMBRYO 
IN  THE  IMBEDDING  MASS,  THE  CONNECTION  WITH  THE  MICROTOME  HOLDER  AND 
THE  ARRANGEMENT  OF  THE  SECTIONS  ON  THE  GLASS  SLIDE. 

Microtome  Holder  The  metal  disc  and  stem  for  holding  the  embryo  in  position 
while  it  is  being  cut. 

Imbedded  Embryo  The  imbedded  embryo  in  the  proper  position  for  sagittal 
Sections. 

Sagittal  Sections     A  slide  of  sagittal  sections  in  the  proper  position  on  the  slide. 

i,  2     Serial  order  in  which  serial  sections  are  arranged  on  the  slide. 

In  section  2,  the  word  sinistral  indicates  that  the  left  surface  of  the  section  faces 
directly  upward.  The  right  side  rests  upon  the  glass. 

The  words  cephalic,  caudal,  dextral  and  sinistral  are  inverted  under  the  com- 
pound microscope,  the  sections  are  reinverted,  and  will  appear  like  this  picture,  if 
the  book  is  turned  upside  down. 

(2)  Mount  the  block  of  paraffin  containing  the  embryo  so  that  the 
right  side  will  be  next  the  disc  of  the  microtome  holder.    The  left  side 
will  then  be  cut  first,  and  look  up  when  mounted  (fig.  242). 

(3)  Place  in  the  microtome  so  that  the  caudal  end  will  first  meet  the 
edge  of  the  knife. 

(4)  Mount  the  sections  in  the  order  of  the  print  on  a  page.     This 
will  bring  the  caudal  end  to  the  left,  the  cephalic  at  the  right,  ventral 
aspect  up  and  dorsal  down  toward  the  lower  edge  of  the  slide.     The 
dextral  face  of  the  section  will  rest  on  the  slide,  and  the  sinistral  face 
will  look  up. 


4io 


MODELS  FROM   SERIAL  SECTIONS 


[Cn.  XI 


Under  the  microscope  the  head  will  be  at  the  left  and  the  dorsal 
side  will  appear  toward  the  upper  edge  of  the  slide  —  away  from  the 
observer.  It  will  appear  like  the  figure  when  the  the  book  is  turned 
upside  down. 

If  the  embryo  is  large  it  may  be  better  to  turn  it  around  so  that 
the  ventral  side  meets  the  edge  of  the  section  knife.  If  this  is  done 
the  sections  will  have  to  be  cut  apart  and  mounted  one  by  one  on  the 


Pifl 


23 


Ss. 


Pig  6 

SI  23 

Sec  253 

10//  260 


1900 


FIG.  243.    SLIDE  OF  SERIAL  SAGITTAL  SECTIONS  SHOWING  THE  ARRANGEMENT 

AND  LABELING. 

slide,  otherwise  they  would  be  crosswise  of  the  slide  like  the  frontal 
sections  (fig.  240). 

§  665.  Labeling  serial  sections.  —  The  label  of  a  slide  on  which 
serial  sections  are  mounted  should  contain  at  least  the  following: 

The  name  of  the  embryo  and  the  number  of  the  series ;  the  number 
of  the  slide  of  that  series;  the  thickness  of  the  sections,  and  the  num- 
ber of  the  first  and  last  section  on  the  slide;  the  date.  It  is  also  a 
convenience  to  have  the  information  repeated  in  part  on  the  left 
end  (fig.  237-243). 

MODELS  FROM  SERIAL  SECTIONS 

§  666.  General  considerations  on  modeling.  —  Anatomists  have  for 
a  long  time  produced  models  of  gross  anatomic  specimens,  and  en- 
larged models  for  minute  details. 

Naturally,  after  serial  sections  of  embryos  and  organs  came  to  be 
made  with  considerable  accuracy  and  of  known  thickness,  there  was  a 
desire  to  make  enlarged  models  which  should  be  exact  representations 
of  the  original  rather  than  the  generalized  approximations  built  up 
as  an  artist  produces  a  statue. 


CH.  XI] 


MODELS  FROM  SERIAL  SECTIONS 


411 


Further,  the  difficulty  of  getting  a  true  conception  of  the  object 
by  studying  only  two  dimensions  in  the  sections  is  very  great;  hence 
a  model  giving  all  three  dimensions  becomes  almost  a  necessity  for 
the  beginner  in  embryology,  and  is  of  enormous  advantage  to  an  in- 
vestigator in  working  out  the  true  form  and  relation  of  complex 
structures.  For  modeling  a  series  it  is  of  great  advantage  to  have 


T- 
H< 

W< 

"  v  I  —  i 

tU  i 

v  , 

r  i 

D- 

n 

D 

A  B 

FIG.  244.     DRYING  OVEN  FOR  SLIDE  TRAYS. 

(From  the  Anatomical  Record). 

A  The  oven  showing  all  the  parts,  the  oven  proper  (j)  is  lifted  up  to  show  the 
electric  lamps  in  the  base  (2). 

B  Sectional  view  of  the  oven  (/)  and  base  (2)  showing  the  construction  and 
the  air  currents.  One  tray  (5)  is  in  position. 

A  The  asbestos  lining  of  the  outer  shell.  B  One  of  the  numerous  ventilating 
holes.  C  Flue  for  the  escape  of  air.  H  Runs  for  the  slide  trays. 

D  Door  of  the  support  or  base  (2).  W-L  Wiring  for  the  lamps.  One  can 
vary  the  heat  by  turning  out  one  or  more  of  the  incandescent  bulbs. 

photographs  of  the  object  to  be  modeled.  If  possible  the  object 
should  be  photographed  in  the  fresh  state  and  after  fixation.  The 
more  aspects  photographed  the  better. 

The  principles  involved  in  the  construction  of  a  model  are  exceed- 
ingly simple:  — 

1.  It  is  necessary  that  the  embryo  or  other  object  to  be  modeled 
should  be  cut  into  a  series  of  sections  of  definite  thickness. 

2.  The  sheets  of  modeling  material  must  be  as  much  thicker  than 
the  sections  as  the  model  is  to  be  larger  than  the  original. 


4i2  MODELS  FROM   SERIAL  SECTIONS  [Cn.  XI 

3.  The  sections  must  be  drawn  as  much  larger  than  the  actual 
specimen  as  the  model  is  to  be  larger  than  the  object. 

4.  The  drawings  with  the  desired  outlines  must  be  made  directly 
upon  or  transferred  to  the  sheets  of  modeling  material  which  are  then 
cut  out,  following  the  lines  of  the  drawing. 

5.  The  different  plates  of  modeling  material  representing  all  the 
sections  are  then  piled  up,  in  order,  thus  giving  an  enlarged  model 
of  the  object  with  all  its  parts  in  proper  position  and  in  true  pro- 
portions. 

MODELS  OF  WAX 

§  667.  Wax  models.  —  For  making  wax  models,  beeswax  820 
grams,  paraffin  270  grams,  and  resin  25  grams  are  melted  together 
and  thoroughly  mixed. 

To  get  the  sheets  of  wax  of  the  proper  thickness  two  methods  are 
available:  — 

(1)  The  hot  wax  is  poured  into  a  vessel  containing  hot  water. 
The  wax  spreads  out  into  an  even  layer  over  the  hot  water  and  is 
allowed  to  cool.     While  it  is  solidifying  it  should  be  cut  free  from 
the  edges  of  the  vessel.     Of  course  by  calculation  and  experiment 
one  can  put  in  the  right  amount  of  wax  to  get  a  plate  of  a  given 
thickness. 

(2)  One  must  have  a  wax-plate  machine  consisting  of  a  flat  sur- 
face —  planed  cast  iron  is  good  —  with  some  means  of  obtaining 
raised  edges.     If  these  are  adjustable  by  a  micrometer  screw  it  is 
simple  to  set  them  properly  for  the  desired  thickness  of  plate.    Then 
there  must  be  a  hot  roller.     The  hot  wax  is  poured  on  the  plate,  and 
with  the  hot  roller  resting  on  the  raised  edges  the  wax  is  rolled  out 
into  a  plate.     It  cools  quickly  and  may  be  removed  for  another  plate. 
This  is  the  most  rapid  and  satisfactory  method  of  preparing  the 
plates.     By  using  a  brush  with  turpentine  the  paper  with  the  draw- 
ing can  be  wet  and  then  with  the  hot  roller  cemented  to  the  plate 
before  that  has  been  removed  from  the  machine. 

The  wax  plate  is  cut  with  a  sharp  instrument,  following  the  outlines 
of  the  object  which  has  been  traced  upon  it  by  the  aid  of  a  camera 
lucida  or  the  projection  microscope.  The  sections  are  piled  together, 
some  line  or  lines  obtained  from  a  drawing  or  photograph  of  the 


CH.  XI] 


MODELS  FROM  SERIAL  SECTIONS 


413 


specimen  before  it  was  imbedded  and  sectioned  being  used  as  a 
guide  by  which  the  correct  form  of  the  pile  of  sections  can  be  tested. 
Finally  the  whole  is  welded  into  one  by  the  use  of  hot  wax  or  a  hot 
instrument.  Models  which  illustrate  complex  internal  structures  are 
difficult  to  prepare,  but  numerous  devices  will  occur  to  the  worker,  as 
the  representation  of  blood  vessels  and  nerves  by  strings  or  wires.  A 
large  model  will  need  much  support  which 
can  be  given  by  wire  gauze,  wires,  pins, 
or  paper,  according  to  the  special  needs. 
A  practical  method  for  wax  modeling 
was  first  published  by  G.  Born,  Arch.  f. 
Mikr.  Anat.,  Bd.  xxii,  1883,  p.  584.  The 
most  detailed  statements  of  improvements 
of  the  method  have  been  published  by 
Born  (Bohn  u.  Oppel),  1904,  and  by  Dr. 
F.  P.  Mall  and  his  assistants.  See  con- 
tributions to  the  Science  of  Medicine,  pp. 
926-1045.  Proceedings  of  the  Amer. 
Assoc.  Anatomists,  1901,  i4th  session 
(1900),  p.  193.  A.  G.  Pohlman,  Zeit.  wiss. 
Mikroskopie,  Bd.  xxiii,  1906,  p.  41. 


FIG.  245.    KINGSBURY'S 
MOVABLE  STAND  FOR  SLIDE 


(From  the  Anatomical 
Record). 


To  overcome  the  difficulty  of  cutting 
out  the  wax  plates,  Dr.  E.  L.  Mark  of 
Harvard  University  uses  an  electrically 
heated  wire  moved  rapidly  by  a  modified    botr^  ^%gresnt  bc"d 
sewing  machine  (Amer.  Acad.  Arts  and        st,  st  Slide  trays. 
Sciences,  March,  1907;  Science,  vol.  xxv,    J^^J^S^ 
1907;  Anat.  Record,  April,  1907).  moved  on  the  floor. 

SUSANNA  PHELPS  GAGE  BLOTTING-PAPER  MODELS 
§  668.  Comparison  of  wax  and  paper  models.  —  Wax  has  certain 
inherent  defects  for  models:  It  is  expensive,  heavy,  and  fragile.  It 
is  easily  deformed  by  the  temperature  of  summer,  and  the  amount  of 
time  necessary  for  the  preparation  of  the  plates  is  great.  A  wax- 
plate  machine  is  expensive  and  bulky. 

It  therefore  seemed  worth  while  to  see  if  there  was  not  some  other 


414  MODELS  FROM  SERIAL  SECTIONS  [Cn.  XI 

material  obtainable  in  the  open  market  which  would  be  more  suit- 
able and  more  generally  available. 

Blotting  paper  seemed  promising,  and  an  actual  trial  showed  it 
to  be  admirably  adapted  for  the  purpose.  Since  making  the  first 
model  in  1905  it  has  been  constantly  used  in  the  laboratory  of  embry- 
ology in  Cornell  University.  Models  made  from  it  were  demon- 
strated before  the  Association  of  American  Anatomists  in  1905  and 
before  the  International  Congress  of  Zoology  in  1907. 

"The  advantages  of  blotting-paper  models  are  the  ease  and  cleanli- 
ness of  their  production  and  the  lightness  and  durability  of  the  prod- 
uct. The  models  are  broken  with  difficulty,  are  easily  packed  or 
transported,  and  when  they  cleave  apart  are  easily  repaired,  thus 
contrasting  with  the  weight  and  fragility  of  wax  models  and  their 
deformation  by  heat." 

"By  this  process  are  secured  for  the  original  model  reconstructed 
from  microscopic  sections  the  same  qualities  which  have  made  the 
Auzoux  models  molded  from  papier-mache  such  useful  and  last- 
ing additions  to  laboratory  equipment;  and,  in  the  hands  of  Dr. 
Dwight  and  Mr.  Emerton,  of  Harvard  University,  have  aided  so 
much  in  the  demonstration  of  structure  and  form  of  special  anatomic 
preparations." 

§  669.  Thickness  of  blotting  paper.  —  Blotting  paper  of  a  uniform 
thickness  of  i  mm.,  T\  mm.,  and  J  mm.  was  found  in  the  market. 
The  i  mm.  is  known  as  140  Ib.  A.  and  costs  about  two  cents  for  a 
sheet  61  X  48  centimeters  (24  x  19  in.). 

The  thickness  is  easily  tested  by  cutting  out  50  small  pieces,  piling 
them,  dipping  one  end  in  melted  paraffin,  and  pressing  them  together. 
The  whole  pile  should  of  course  measure  50  mm.  if  the  paper  is  milli- 
meter paper  (§  669a). 

§  669a.  —  Book-stores,  paper  dealers  and  job  printers  are  supplied  by  the 
paper  manufacturers  with  samples  of  blotting  paper.  One  can  look  these 
samples  over,  select  and  order  the  kinds  desired.  The  millimeter  blotting 
paper  mentioned  in  the  text  is  one  of  the  cheaper  grades,  costing  by  the  pack- 
age of  500  sheets  about  two  cents  a  sheet  (sheets  61  x  48  centimeters,  24  x  19 
inches). 

§  670.  Size  of  the  model.  —  In  deciding  upon  the  size  of  the  model 
to  be  made  from  a  given  series  of  sections  one  should  select  the  largest 


CH.  XI]  MODELS  FROM  SERIAL  SECTIONS  415 

section  and  with  the  projection  microscope  throw  the  image  on  the 
table  (fig.  246).  By  using  different  objectives  and  different  distances 
from  the  microscope  one  can  find  a  size  which  seems  suitable.  The 
magnification  may  be  found  by  §  276.  Then  by  multiplying  the  whole 
number  of  sections  by  the  thickness  of  the  sections  and  this  by  the 
magnification',  one  can  get  the  length  or  height  of  the  model.  One 
must  take  these  preliminary  steps  and  decide  upon  the  magnification 
to  be  used  or  the  model  is  liable  to  be  too  large  to  be  manageable  or 
too  small  to  show  well  the  necessary  detail. 

(1)  Suppose  the  model  is  to  be  100  times  the  size  of  the  original 
object,  and  the  object  has  been  cut  into  a  series  of  sections  IOJJL  thick. 
Then  each  section  must  be  represented  by  a  plate  or  sheet  100  times 
as  long,  broad,  and  thick  as  the  object.     As  the  sheets  of  blotting 
paper  are  so  large  (61  X  48  cm.),  one  need  be  solicitous  only  about 
the  thickness. 

As  each  section  is  actually  IOJJL  thick  and  the  model  is  to  be  100 
times  enlarged,  the  thickness  representing  each  section  must  be 
ioju  X  ioo  =  loooju,  or  i  millimeter,  i  millimeter  blotting  paper  is 
used  and  every  section  of  the  series  is  drawn. 

(2)  If  the  blotting  paper  were  only  fV  mm.  thick  it  would  be 
simpler  to  make  the  model  90  times  the  size  of  the  original.     If,  how- 
ever, one  wished  the  magnification  to  be  ioo,  it  could  be  accomplished 
thus:   Each  section  in  the  series  should  be  represented  by  i  mm.  or 
loooju  in  thickness.     But  if  one  uses  blotting  paper  of  •&•  mm.  thick- 
ness or  9ooju,  there  is  a  loss  of  ioo/i  for  each  section  and  for  9  sections 
there  would  be  a  loss  of  900/1,  or  the  thickness  of  a  sheet  of  the  blotting 
paper.     To  remedy  this  one  uses  10  sheets  of  blotting  paper  for  9 
sections.     This  keeps  the  model  in  true  proportion.     In  practice  each 
of  the  sections  is  drawn  upon  one  sheet  except  one  of  them  and  for 
that  two  sheets  of  the  blotting  paper  are  united  and  the  sections 
drawn  upon  the  double  sheet. 

§  671.  General  rule  for  the  use  of  blotting  paper.  —  Divide  the 
thickness  by  which  each  section  is  to  be  represented  in  the  model  by 
the  thickness  of  one  sheet  of  the  blotting  paper  available.  The  quo- 
tient shows  the  number  of  sheets  or  the  fraction  of  a  sheet  required 
for  each  section. 


4i6  MODELS  FROM  SERIAL  SECTIONS  [Cn.  XI 

If  a  quotient  is  a  mixed  number  reduce  it  to  a  fraction.  The 
numerator  represents  the  number  of  sheets  required  and  the  denomi- 
nator the  number  of  sections  to  go  with  the  sheets. 

Examples:  (a)  With  a  series  of  loju  sections  to  be  modeled  at  100 
enlargement  each  section  of  the  series  must  be  represented  in  the 
model  by  a  thickness  of  10/4  x  100  =  1000/4  or  i  millimeter.  If  one 
uses  millimeter  or  IOOOJJL  paper,  then  1000/4  -f-  IOOO/A  =  -J-,  and  one 
must  use  i  sheet  for  i  section. 

(b)  With  a  series  of  10/4  sections  to  be  made  into  a  model  100  times 
enlarged,  and  with  blotting  paper  of  •&  mm.  or  900/4  thickness,  each 
section  must  be  represented  by  10/4  x  100  =  1000/4.     If  the  blotting 
paper  is  900/4  thick,  then  it  requires  for  each  section:   1000  -f-  900  =  i^ 
sheets  of  paper  or  -^  sheets  for  one  section  or  10  sheets  for  9  sections, 
that  is  a  double  sheet  for  one  of  the  nine  sections. 

(c)  With  a  series  cut   15/4,  for  a  50  fold  model,   each  section  is 
represented  by  a  thickness  of  15/4  X  50  =  750/4.     If  one  uses  i  mm. 
or  looo/t  blotting  paper,  then  each  section  requires  750  -f-  1000/4  =  J 
of  a  sheet  for  one  or  3  sheets  for  four  sections.     In  this  case  one 
omits  every  fourth  section  in  drawing,  thus:   ist,  2d,  and  3d  sections 
would  be  drawn;   then  the  5th,  6th  and  7th;    gth,  loth,  nth,  etc., 
every  fourth  being  omitted. 

(d)  If  for  the  model  just  considered  one  had  y9<j  mm.  or  900/4  paper 
then  750  -i-  900  =  f .    That  is  there  must  be  5  sheets  of  the  paper  for 
each  6  sections.    In  that  case  every  sixth  section  would  be  omitted 
in  the  drawing,  as  every  fourth  section  was  omitted  in  (c). 

It  is  of  course  best  to  use  sheets  of  exactly  the  right  thickness  to 
represent  the  necessary  thickness  in  the  model  (a),  but  one  can  pro- 
duce models  with  accuracy  by  duplicating  one  or  more  sheets  for 
a  group  of  sections  (b)  or  by  omitting  certain  sections  of  the  series 
in  drawing  (c,  d). 

DRAWINGS  FOR  MODELS 

§  672.  —  The  methods  given  for  drawing  microscopic  preparations 
in  Ch.  VI  are  all  applicable  except  the  freehand  method.  This  is  not 
applicable,  because  it  is  not  possible  to  draw  at  a  uniform  and  accurate 
enlargement  in  that  way.  But  the  camera  lucida  method  (§  275) 


CH.  XI]  MODELS  FROM   SERIAL  SECTIONS  417 

or  the  projection  apparatus  method  (§  293)  is  good.  With  the  per- 
fecting of  projection  apparatus  that  method  is  far  the  best  because 
one  can  sit  in  a  comfortable  position  and  use  both  eyes.  It  is  indeed 
as  simple  as  tracing  the  outline  of  actual  pictures. 

By  making  negative  prints  directly  on  one  of  the  developing  papers 
(§  363),  drawing  for  models  may  be  wholly  avoided. 

§  673.  Avoidance  of  distortion  and  of  inversion.  —  In  the  drawings 
for  models  one  must  of  course  avoid  all  distortion  (§269)  and  the 
inversion  of  the  image  (§277).  Both  these  defects  are  easily  avoided 
if  one  keeps  in  mind  the  optical  principles  involved,  and  follows  the 
directions  given  in  Ch.  VI. 

§  674.  Use  of  the  6-volt,  concentrated  filament  lamp  as  a  source 
of  light.  —  From  the  experience  of  the  author  nothing  equals  the 
direct-current  arc  light  for  all  exacting  work  in  drawing  and  projec- 
tion, and  for  the  dark-ground  illuminator,  but  the  care  required  to 
keep  the  arc  lamp  going  and  to  keep  the  crater  centered  is  so  great 
that  the  less  brilliant  light  from  the  6-volt  lamp  which  requires  abso- 
lutely no  adjustment  after  being  once  properly  arranged  is  very 
acceptable  (§  362).  The  6-volt  lamp  with  a  transformer  is  used  only 
on  an  alternating  circuit.  As  most  lighting  circuits  are  now  alternat- 
ing it  is  a  great  advantage;  and  as  this  lamp  with  its  transformer 
can  be  used  anywhere  wherever  there  is  an  ordinary  electric  light 
socket,  it  is  exceedingly  convenient.  If  it  is  to  be  used  on  a  direct 
current  circuit  no  transformer  is  used,  but  the  current  must  be  drawn 
from  a  storage  battery,  not  from  a  no  or  a  220  volt  circuit  from  a 
dynamo. 

§  675.  Connections  of  the  transformer.  —  If  alternating  current 
and  a  transformer  are  used,  the  transformer  must  be  connected  to 
the  supply  by  means  of  the  small  connecting  wires.  The  connection 
with  the  lamp  is  by  the  large  terminal  wires.  Ordinarily  the  terminals 
of  the  transformer  are  marked  so  that  no  mistake  need  be  made. 
Theoretically  the  transformer  does  not  modify  the  energy;  it  either 
raises  or  lowers  the  voltage  or  pressure.  For  the  purposes  here  used 
the  transformer  lowers  the  voltage,  and  is  called  a  "step  down  trans- 
former." As  the  activity  or  wattage  of  which  the  current  is  capable 
is  not  changed  by  the  transformer,  and  as  the  wattage  is  the  voltage 


4i8 


MODELS  FROM   SERIAL  SECTIONS 


[Cn.  XI 


multiplied  by  the  amperage  used,  if  the  voltage  is  lowered  the  amperage 
is  raised  proportionally;  hence  the  need  of  the  large  wire  on  the  side 
toward  the  lamp  beyond  the  transformer  where  the  amperage  is 
increased. 

§  676.  Lamp  for  6-volt  current.  —  There  are  in  common  use  two 
lamps,  one  of  72  watts  and  one  of  108  watts.  Now  as  the  wattage 
is  the  voltage  times  the  amperage,  for  the  7  2 -watt  lamp  the  amperage 
^  Lamp  with  a  6-volt  current  must 

^-•^  be  72  divided  by  6  or  12 
amperes.  For  the  108- 
watt  lamp  in  like  manner 
the  amperage  is  the  watt- 
age divided  by  the  volt- 
age, —  1 08  divided  by  6  = 
1 8  amperes.  *  This  shows 
at  once  why  the  large 
wires  must  be  used  be- 
tween the  lamp  and  the 
transformer.  If  the  usual 
small  wires  are  used  the 
resistance  is  too  great 
and  part  of  the  energy  is 
used  up  in  heating  the 


FIG.  246.  DRAWING  AND  PROJECTION  OUTFIT 
WITH  LARGE  MIRROR  ON  SEPARATE  DRAWING 
TABLE. 

For  full  explanation  see  Fig.  112.  Instead  of 
the  arc  lamp  here  shown  the  6-volt  incandescent 
lamp  can  be  used  for  most  purposes  (§676). 


wires  instead  of  in  heating  the  filament  to  supply  the  light. 

It  is  also  a  good  plan  to  have  the  wires  between  the  transformer  and 
the  lamp  as  short  as  possible  and  not  be  inconvenient. 

§  677.  Arrangement  of  the  lamp  for  the  large  projection  outfit.  - 
If  the  lamp  is  to  be  used  in  the  lamp-house  instead  of  an  arc  lamp 
for  the  large  projection  outfit,  it  must  be  centered  carefully  and  put 
the  right  distance  from  the  large  condenser.  The  filament  takes  the 
place  of  the  crater  of  the  arc  lamp  and  hence  should  be  in  the  focus 
of  the  first  element  of  the  condenser,  so  that  the  beam  between  the  first 
and  second  elements  of  the  condenser  will  be  approximately  parallel. 

If  a  two-lens  condenser  is  used  the  lamp  filament  is  slightly  within 
the  focus,  making  the  light  slightly  diverging  between  the  two  lenses 
of  the  condenser. 


CH.  XI]  MODELS  FROM  SERIAL  SECTIONS  419 

A  concave  mirror  or  reflector  behind  the  lamp  is  of  considerable 
advantage,  for  the  light  which  extends  backward  is  reflected  forwjard 
to  the  condenser  and  is  thus  available  for  illuminating  the  object. 

§  678.  Large  condenser  for  drawing.  —  If  the  three-lens  condenser 
is  used  (fig.  in),  and  it  is  much  to  be  preferred,  the  second  element 
which  converges  the  parallel  beam  should  be  of  long  focus.  One  of 
38  cm.  (15  in.)  focus  has  been  found  very  satisfactory.  The  reason 
for  using  the  long  focus  lens  is  discussed  in  Ch.  VI,  §  297,  fig.  115. 

If  a  two-lens  condenser  is  used  the  second  element  should  also  be 
of  longer  focus  than  for  ordinary  magic  lantern  work,  for  the  same 
reason  as  for  the  three-lens  condenser. 

§  679.  Drawing  with  the  small  projection  outfit.  —  If  one  has  no 
large  projection  outfit,  drawings  for  models  and  for  publication  can 
be  made  very  satisfactorily  with  the  6-volt  lamp  as  follows:  It  is 
a  great  advantage  to  have  the  lamp  in  one  of  the  metal  lanterns 
like  those  used  for  daylight  glass  (fig.  37-38),  then  scattered  light  will 
be  avoided.  There  should  be  a  condenser  like  that  used  for  the  small 
arc  lamp  (fig.  49).  It  should  be  over  one  of  the  daylight  glass  open- 
ings and  of  course  centered  with  the  lamp  filament.  If  it  were  in  a 
tube  which  permitted  of  a  limited  amount  of  movement,  as  with  the 
condenser  of  the  small  arc  lamp,  it  would  be  of  much  advantage. 
As  the  microscope  must  be  horizontal  and  is  ordinarily  raised  to 
make  the  drawing  distance  250  mm.,  the  lantern  containing  the  6-volt 
lamp  must  be  supported  on  a  box  or  block  to  bring  the  filament  of 
the  lamp  in  the  optic  axis  of  the  microscope. 

When  horizontal  the  microscope  is  unstable;  hence  a  weight  or 
better  a  clamp  is  put  over  the  feet  to  hold  the  microscope  firmly 
so  that  when  once  centered  it  will  not  move  easily.  A  table  with 
the  drawing  shelf  on  the  legs  is  very  convenient  for  getting  the  de- 
sired magnification  (fig.  247). 

§  680.  Relative  position  of  the  lamp  and  microscope.  —  This  can 
be  as  with  the  small  drawing  outfit  and  arc  lamp  (fig.  113),  or  it  can 
be  put  in  line,  as  with  the  large  outfit.  If  in  line  (fig.  in)  the  mirror 
is  not  used,  and  care  must  be  taken  to  get  all  parts  lined  up  to  one 
axis.  With  the  mirror  slight  deviations  from  centering  can  be  over- 
come by  inclining  the  mirror  accordingly. 


420 


MODELS   FROM   SERIAL  SECTIONS 


[On.  XI 


§  681.  Condensers  to  use  with  the  small  outfit.  —  For  low  powers, 
50. to  1 6  mm.,  the  substage  condenser  of  the  microscope  can  be  turned 
aside  and  the  small  condenser  with  the  lamp  alone  employed.  In 
many  cases  no  ocular  is  used  for  the  sake  of  the  large  field.  For  powers 
of  8  to  2  mm.  when  the  ocular  is  used  it  is  necessary  to  use  the  sub- 
stage  condenser  to  light  with  the  proper  aperture.  And  if  the  oil 
immersion  is  used  it  is  a  great  advantage  to  make  the  substage  con- 


Microscope 


Condenser 


DRAWING  AND  PROJECTION  OUTFIT. 


For  full  explanation  see  Fig.  109.  For  drawing  the  6-volt  lamp  can  well  take 
the  place  of  the  arc  lamp  here  shown  (§676). 

denser  homogeneous  immersion  also;  that  is,  to  have  some  of  the 
homogeneous  immersion  fluid  ^between  the  lower  side  of  the  slide 
and  the  condenser  as  well  as  between  the  objective  and  the  cover- 
glass  (§471)- 

§  682.  Making  the  drawings.  —  One  can  draw  directly  upon  blot- 
ting paper,  but  it  is  so  important  to  have  a  drawing  to  refer  back  to 
that  one  or  more  duplicates  should  be  made.  This  is  easily  accom- 
plished by  putting  a  sheet  of  carbon  manifolding  paper  on  the  blot- 
ting paper  and  a  sheet  of  thin  paper  over  the  carbon  paper,  using 
thumb-tacks  to  hold  the  blotting  paper  and  the  duplicating  sheets 
in  position. 


CH.  XI]  MODELS  FROM   SERIAL  SECTIONS  421 

One  should  take  the  precaution  to  number  each  drawing  as 
it  is  made;  then  confusion  in  the  later  processes  will  be 
avoided. 

§  683.  Cutting  out  the  sheets  for  the  model.  —  "With  the  blotting 
paper,  if  the  drawings  are  small  the  cutting  is  easily  done  with  scissors 
or  a  knife.  When  the  drawings  are  large  and  especially  when  the 
model  is  to  be  made  by  representing  each  section  by  two  or  more 
thicknesses  of  blotting  paper,  it  has  been  found  that  an  ordinary 
sewing  machine  can  be  used  to  do  the  cutting.  By  setting  the  regu- 
lator for  the  shortest  stitch,  an  almost  continuous  cut  is  made  and 
the  parts  are  easily  separated.  If  a  large  sewing-machine  needle  is 
sharpened  in  the  form  of  a  chisel,  the  cut  becomes  considerably 
smoother.  It  has  been  found  advantageous  when  long  continued  or 
heavy  work  is  to  be  done  to  attach  to  the  machine  an  electric  sewing- 
machine  motor.  Skill  in  guiding  the  work  is  soon  acquired.  There 
are  some  details  of  a  complicated  drawing  which  are  more  easily  cut 
by  the  scissors  or  a  knife  after  the  main  lines  have  been  cut  by  the 
machine." 

§  684.  Contrasting  colors  for  marking  groups  of  sections.  —  "It  is 
a  great  advantage  in  any  working  model  to  have  sections  at  regular 
intervals  in  marked  contrast  with  the  body  of  the  material.  Blot- 
ting paper  of  a  large  variety  of  colors  (black,  red,  blue,  pink)  is  easily 
obtained  in  the  market.  In  the  models  made  every  tenth  plate  was 
a  bright  or  light  color  and  every  one-hundredth  was  black,  rendering 
rapid  numeration  easy." 

§  685.  Putting  the  sheets  together  to  make  the  model.  -  "When 
the  paper  sections  are  thus  prepared  they  are  piled  and  repiled  as  is 
usual  until  the  shape  conforms  to  an  outline  predetermined  from 
photographs,  drawings,  or  measurements  made  before  the  specimen 
was  cut." 

"It  has  been  found  that  an  easily  prepared  support  and  guide  for 
the  model  in  process  of  setting  up  is  made  by  cutting  the  outline 
to  be  followed  from  a  block  of  four  or  five  sheets  of  blotting  paper, 
marking  upon  it  the  lines  of  direction  of  every  tenth  or  twentieth 
section.  The  colored  numerating  plates  must  of  course  conform  to 
the  spacing  and  direction  of  these  lines." 


422  MODELS   FROM   SERIAL  SECTIONS  [Cn.  XI 

i  "The  preliminary  shaping  having  been  accomplished,  more  exact 
modeling  is  undertaken.  The  paper  sections  slide  very  easily  upon 
one  another.  The  most  satisfactory  means  of  fastening  them  to- 
gether is  by  the  use  of  ribbon  pins,  ordinary  pins,  or  wire  nails  of 
various  sizes,  depending  on  the  size  of  the  model.  No  kind  of  paste 
or  glue  was  found  suitable  for  this  purpose." 

§686.  Finishing  the  model.  —  "When  the  model  is  well  formed, 
inequalities  are  best  removed  by  rubbing  with  the  edge  of  a  dull 
knife  and  smoothing  with  sandpaper.  Any  dissections  of  the  model 
for  showing  internal  structures  should  be  planned  for  at  this  stage, 
for  it  is  now  more  easily  separated  than  later.  It  is  also  at  this  tune 
that  superfluous  'bridges,'  which  have  been  left  in  place  to  support 
detached  parts,  would  better  be  removed." 

"To  finish  the  model  it  is  held  together  firmly  and  coated  with 
hot  paraffin  either  by  a  camel's  hair  brush  or  by  dipping  in  paraffin 
and  removing  the  superfluous  coating  by  a  hot  instrument.  One 
might  use  a  thermo-cautery  for  this  purpose." 

"The  paraffin  renders  the  model  almost  of  the  toughness  of  wood 
without  destroying  the  lightness  of  the  paper." 

§  687.  Coloring  the  surface;  dissecting  the  model.  —  "For  color- 
ing the  surface  of  the  model,  it  was  found  most  desirable  to  use 
Japanese  bibulous  paper,  lens  paper  (§  158)  which  had  been  dipped 
in  water  color  and  dried.  Any  of  the  laboratory  dyes  or  inks  can 
be  used,  such  as  eosin,  picric  acid, ,  methylene  green,  black  ink, 
etc.  The  colored  lens  paper  molds  over  the  surface  with  ease  and 
is  held  in  place*  by  painting  with  hot  paraffin.  All  color  and 
enumeration  lines  and  fine  modeling  show  through  the  transparent 
paper." 

"When  the  model  ceases  to  be  a  working  model  it  can  be  covered 
with  oil  paints  mixed  with  hot  paraffin  and  rubbed  to  any  degree  of 
finish  desired." 

"One  can  dissect  a  model  by  a  hot  knife  run  along  the  planes 
of  cleavage  or  cut  across  them  by  a  saw." 

For  the  literature  of  blotting  models  see:  Susanna  Phelps  Gage, 
Amer.  Jour.  Anat.,  vol.  v,  1906,  p.  xxiii;  Proceedings  of  the  Inter- 
national Zoological  Congress  for  1907;  Anatomical  Record,  Nov. 


MODELS  FROM  SERIAL  SECTIONS  423 

1907.     (From  this  paper  the  above  quotations  were  made.)    Zeit. 
wiss.  Mikroskopie,  Bd.  xxv,  1908,  pp.  73-75. 

Blotting-paper  models  have  also  been  made  and  demonstrated 
by  Dr.  J.  H.  Hathaway  and  by  Dr.  J.  B.  Johnston  at  the  Association 
of  American  Anatomists,  1906  (Proc.  Assoc.  Amer.  Anatomists,  Anat. 
Record,  April  i,  1907);  in  1909  by  Dr.  J.  Parsons  Schaeffer  (Anat. 
Record,  1910);  and  in  1916  by  Dr.  Charles  Brookover  and  Dr. 
H.  Saxon  Burr  (Anat.  Record,  1917). 


CHAPTER  XII 
BRIEF   HISTORY    OF   LENSES   AND    MICROSCOPES 

IN  works  and  papers  dealing  with  the  history  of  the  microscope,  it 
seems  to  the  writer  that  undue  prominence  is  given  to  the  mere  me- 
chanical supports  and  arrangements  for  focusing  the  optical  parts. 
These  were  legion,  and  they  are  being  improved  year  by  year  even 
faster  than  the  optical  parts.  The  mechanical  parts  are  not  to  be 
belittled,  but  after  all  it  is  the  optical  parts  that  make  a  microscope, 
and  some  of  the  most  fundamental  work  of  the  world  in  the  microscopi- 
cal field  has  been  accomplished  with  instruments  which  now  seem  very 
unattractive  mechanically. 

The  mechanical  parts  of  the  microscope  have  been  figured  and 
described  fully  in  the  Journal  of  the  Royal  Microscopical  Society; 
in  Harting's  work  on  the  microscope,  and  in  the  histories  of  Mayall 
and  Petri.1 

It  is  hoped  that  by  dealing  with  the  optical  parts  only  the  reader 
will  gain  a  connected  and  comprehensive  view  of  the  main  steps  which 
have  been  taken  in  bringing  about  the  optical  instruments  of  the 
present  day. 

§  690.  Lenses.  —  It  is  difficult  to  think  of  a  world  without  lenses. 
All  apparatus  like  the  moving  picture  machine,  magic  lantern,  photo- 
graphic camera,  the  microscope  and  telescope  and  spectacles,  would 
be  no  more.  But  it  is  not  to  be  forgotten  that  the  most  splendid 
creations  in  the  world  of  art,  as  that  of  the  Greeks;  and  in  the  world 
of  literature,  as  that  of  the  Hebrews,  the  Greeks,  and  the  Romans; 
the  architecture  of  the  Orient,  of  Egypt,  Greece  and  Rome;  and  the 
feats  of  engineering  of  the  ancient  world  were  all  independent  of 

1  The  author  wishes  to  express  his  appreciation  of  the  help  given  by  Dr.  A.  C. 
White,  of  the  Cornell  University  Library,  in  translating  passages  from  the  Greek 
and  Latin  works  bearing  upon  optics  and  vision. 

424 


CH.  XIi:          HISTORY  OF  LENSES  AND  MICROSCOPES  425 

lenses  and  the  optical  instruments  which  they  make  possible.  But 
what  immeasurably  greater  insight  into  the  real  world  has  come  with 
these  "  optic  glasses" !  What  revelations  as  to  the  cause  of  disease, 
of  the  structure  of  the  universe  in  its  smallest  details  by  the  micro- 
scope, and  in  its  larger  ranges  by  the  telescope;  and  greatest  of  all 
for  the  common  man,  has  come  the  power,  by  means  of  spectacles,  to 
make  good  use  of  the  years  that  hygiene  has  added  to  the  average 
human  life. 

That  nature  made  lenses  during  every  rain-storm  and  every  heavy 
dew  and  in  the  tears  of  every  gum  and  balsam  tree,  wre  know  now; 
and  for  the  almost  infinite  years  which  man  has  been  upon  the  earth, 
the  learned  and  the  ignorant  were  equally  unmindful  of  the  marvel 
before  their  very  eyes;  as  unmindful  as  are  the  vast  majority  of  men 
and  women  at  the  present  day. 

All  who  have  made  a  study  of  the  question  are  unanimous  in  the 
opinion  that  optical  instruments,  other  than  mirrors,  were  unknown 
to  the  ancient  world;  and  that  lenses  were  wholly  unknown.  Some, 
however,  find  in  the  disc  of  quartz  in  the  British  Museum  and  known 
as  the  Assyrian  "  lens,"  and  dating  from  about  700  B.C.,  evidence 
that  lenses  were  made  before  the  Christian  era.  How  one  who  actually 
sees  this  disc  of  quartz  can  think  of  it  as  a  lens  is  inconceivable  to  me. 
Mayall,  who  had  an  opportunity  to  study  it,  decides  wholly  against 
the  lens  theory.  In  his  work  on  the  history  of  the  microscope,  p.  5, 
he  gives  a  face  and  an  edge  view  of  it. 

In  the  first  and  second  centuries  of  the  Christian  era  there  was  an 
abundance  of  knowledge  of  mathematics  and  of  optics  to  make  possi- 
ble the  invention  of  the  simple  microscope  and  of  appreciating  it  as 
such.  In  works  of  literature  there  are  hints  that  men  were  on  the 
track.  For  example,  Seneca,  in  his  Questiones  Naturales  (L.  i,  q.  6), 
says  that  "  Letters  however  small  and  dim  are  comparatively  large 
and  distinct  when  seen  through  a  glass  globe  filled  with  water,"  and 
that  apples  in  a  vase  of  water  are  far  more  beautiful.  He  is  trying  to 
account  for  the  size  of  the  rainbow  and  sums  it  all  up  by  saying  that, 
"  anything,  in  fact,  that  is  seen  through  moisture  appears  far  larger 
than  in  reality  it  is."  To  Seneca  the  magnification  was  the  effect  of 
the  water  and  not  the  effect  of  the  refraction  at  curved  surfaces, 


426  HISTORY  OF  LENSES  AND  MICROSCOPES          [Cn.  XII 

Turning  now  from  the  literature  of  this  period  to  the  work  of  Clau- 
dius Ptolemaeus  (70-147  A.D.)  on  optics,  one  is  filled  with  admira- 
tion for  the  exactness  of  knowledge  displayed.  He  stated  with  a 
clearness  never  since  excelled  the  laws  of  refraction  of  light  in  passing 
from  transparent  media  of  different  density,  and  dealt  with  curved 
as  well  as  with  plane  surfaces  (Sermo  Quintus).  It  is  almost  incon- 
ceivable that  he  should  not  have  discovered  the  magnifying  power  of 
curved  bodies  from  then*  refractive  action.  A  part  of  this  discussion 
is  lost,  but  so  far  as  known  he  did  not  make  that  discovery. 

In  works  dealing  with  the  history  of  optics  frequent  reference  is 
made  to  Alhazen  "  On  Appearances."  This  work  is  supposed  to  date 
from  about  noo  A.D.  It  was  translated  from  the  Arabic  by  Risner 
in  1572.  Almost  all  of  its  sound  teaching  in  optics  conforms  very 
closely  with  that  of  Ptolemaeus  whom  Alhazen  mentions.  The  struc- 
ture and  action  of  the  eye  is  founded  almost  entirely  on  the  work  of 
Galen.  In  using  Alhazen  one  should  note  carefully  what  is  said  by 
Risner  in  the  preface,  for  it  seems  quite  possible  from  his  statement 
that  he  might  unconsciously  have  read  into  his  translation  knowledge 
of  optics  which  was  a  later  acquisition;  in  a  word  in  trying  to  make 
clear  the  work  of  Alhazen,  possibly  a  certain  amount  of  later  knowledge 
was  added  to  it. 

In  passing  it  may  be  said  in  reading  almost  any  of  the  ancient 
writers  and  indeed  all  large  publications  of  a  single  author,  that  they 
are  in  the  nature  of  cyclopedias,  detailing  the  knowledge  most  in  favor 
at  the  time  and  often  containing  a  certain  amount  of  original  matter. 
The  older  the  work  the  greater  the  proportion  of  original  matter  if 
the  author  is  of  first  rate  ability,  because  until  recently  there  have 
not  been  the  periodicals  and  transactions  of  learned  societies  in  which 
to  publish  one's  original  contributions. 

The  first  clear  and  unmistakable  statements  from  which  dates 
modern  knowledge  of  lenses  and  their  action  are  found  in  the  works 
of  Roger  Bacon;  especially  his  Opus  Majus,  1266-1267.  Roger 
Bacon's  work  is  encyclopedic  in  many  ways,  and  in  many  it  is  like 
a  modern  monograph,  giving  full  recognition  of  the  opinions  and  work 
of  others.  In  his  works  (Opus  Majus;  Opus  Tertium,  etc.)  lenses  are 
figured  and  discussed  in  detail.  Bacon  nowhere  claims  to  be  the 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES  427 

inventor  of  lenses.  He  expounds  the  principles  on  which  they  act, 
referring  back  to  Ptolemaeus  for  the  laws  of  refraction  so  clearly  set 
forth  by  him.  And  he  discusses  over  and  over  again  the  marvelous 
things  which  lenses  enable  one  to  do.  Nearly  all  of  the  things  men- 
tioned by  Bacon  we  know  are  possible  from  our  own  experience. 
He  tells  us  that  much  of  his  private  fortune  was  spent  in  obtaining 
apparatus  of  all  kinds,  for  he  insisted  that  the  final  test  in  science 
is  experiment. 

He  pointed  out  that  convex  lenses  made  it  possible  for  old  men 
to  read  the  smallest  letters,  and  within  thirty-two  years  from  that 
time,  i.e.,  in  1299,  we  have  in  a  manuscript  this  notable  sentence: 
"  I  am  so  affected  by  years  that  I  cannot  read  or  write  without  the 
glasses  they  call  spectacles,  lately  found  out  for  the  benefit  of  old 
men  when  their  eyesight  gets  weak"  (Carpenter-Dallinger,  p.  118, 
Harting,  III,  p.  16). 

§  691.  Spectacles.  —  It  is  rather  surprising  that  the  use  of  spec- 
tacles became  so  general  in  so  short  a  time  after  Bacon  had  sent  his 
manuscript  to  Pope  Clement  IV.  The  part  on  optics  (Perspectiva) 
was  copied  many  times  and  widely  distributed  among  the  libraries. 
It  is  referred  to  by  many  writers,  e.g.  Porta,  Maurolycus,  Kepler, 
Scheiner,  etc.  Apparently,  then,  it  was  available  for  any  one  who  was 
interested  greatly  in  optics.  For  over  300  years  from  the  tune  of 
Roger  Bacon  practically  all  of  the  work  in  optics  was  in  the  hands  of 
the  spectacle  makers,  and  to  them  we  owe  both  the  telescope  and  the 
microscope  and  the  lenses  for  the  camera  obscura  and  the  magic 
lantern  (§  701-704). 

§  692.  Concave  spectacles.  —  Roger  Bacon  knew  concave  as  well  as 
convex  lenses,  but  he  did  not  refer  to  the  use  of  concave  lenses  for 
people  with  short  sight  (myopia)  so  far  as  I  have  been  able  to  find  out. 
Just  who  made  this  discovery  has  never  been  shown.  However, 
within  200  years  from  the  first  statements  in  the  Opus  Majus  con- 
cerning the  use  of  convex  glasses  for  old  men,  mention  becomes  more 
and  more  common  of  concave  glasses  for  the  myopes,  and  from 
1568  onward  convex  and  concave  spectacles  have  a  fixed  place 
as  aids  to  vision.  See  Barbaro,  §  7osa,  and  the  works  of  Pansier, 
p.  20-31,  and  Bock,  p.  44. 


428  HISTORY  OF  LENSES  AND  MICROSCOPES          [Cn.  XII 

§  693.  Cylindrical  spectacles  —  astigmatism.  —  The  use  of  specta- 
cles for  defective  vision  was,  until  quite  recently,  confined  to  those 
with  long  sight  or  short  sight,  and  spherical  convex  or  concave  lenses 
were  used.  Two  English  astronomers  and  physicists  (Thomas  Young. 
1800,  and  George  B.  Airy,  1825)  found  that  the  curvature  in  their 
eyes  were  not  equal  and  consequently  lines  in  one  plane  focused  at 
one  level  and  in  another  plane  at  a  different  level.  To  correct  this 
defect  Young  pointed  out  that  the  spectacles  might  be  tilted,  and 
Airy  that  the  best  way  was  to  use  cylindrical  glasses  which  would 
just  neutralize  the  unequal  curvature.  Probably  this  discovery  of 
astigmatism  and  the  means  for  its  correction  has  and  is  destined  to 
accomplish  greater  good  to  the  human  race  than  any  other  optical 
device  of  the  igth  century. 

Some  little  space  has  been  devoted  to  spectacles  because  the  eyes 
are  a  fundamental  part  of  any  optical  combination  like  the  microscope 
or  telescope  and  is  the  judge  of  the  real  images  produced  by  any  optical 
train  like  the  photographic  camera,  the  magic  lantern  and  the  mov- 
ing picture  machine,  therefore  whatever  pertains  to  the  eye  and  its 
natural  perfection  or  artificial  means  of  making  it  more  perfect,  is 
germane  to  the  subject. 

§  694.  Simple  microscope.  —  Every  convex  lens  is  or  may  be  used 
as  a  microscope,  as  it  aids  the  eye  in  seeing  an  object  under  an  in- 
creased visual  angle,  and  hence  makes  it  appear  larger  than  it  would 
if  viewed  by  the  naked  eye.  Hence,  when  considering  the  history  of 
the  simple  microscope  it  is  evident  that  that  history  is  the  same  as 
the  history  of  convex  lenses.  The  date  of  the  invention  is  some  time 
before  the  date  of  the  Opus  Majus  of  Roger  Bacon.  He  speaks  of 
them,  not  as  a  wholly  new  invention  of  his  own  time,  but  as  one 
of  the  means  by  which  wonderful  things  can  be  done.  His  whole 
purpose  in  the  discussion  was  to  induce  the  church  to  make  the 
fullest  use  of  all  the  products  of  science  to  give  the  superiority  which 
he  felt  was  the  right  and  the  privilege  of  the  Christian  world  to 
possess  in  its  efforts  for  advancing  civilization. 

The  simple  lens  or  the  combination  of  lenses  making  up  a  simple 
microscope  may  be  held  in  the  hand,  but  ordinarily  there  is  some 
metal  binding  and  support  for  the  protection  of  the  lens  or  lenses,  and 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES 


429 


their  easier  handling  or  focusing.  The,  common  reading  glass  with 
its  convenient  handle  (fig.  4)  and  the  tripod  (fig.  201)  and  focusing 
lens  holder  (fig.  202)  are  good  examples. 

In  reading  the  older  literature  one  often  meets  with  the  expression 
"  single  microscope."  This  means  a  simple  microscope,  composed  of 
one  lens  (fig.  182),  and  is  in  contrast  with  the  "  double  microscope," 
or  compound  microscope  of  two  lenses  or  two  combinations  (objective 
and  ocular,  fig.  248-249). 


Ocular 


FIG.  248,  249. 


DUTCH  AND  KEPLERIAN  COMPOUND  MICROSCOPES  FOR 
COMPARISON. 


Each  has  a  convex  lens  for  objective.  For  ocular  the  Dutch  form  has  a  concave 
and  the  Keplerian  farm  a  convex  lens.  The  ocular  for  the  Keplerian  form  is 
properly  a  magnifier  of  the  real  image,  while  the  concave-lens  ocular  of  the  Dutch 
microscope  acts  as  an  amplifier  for  the  objective. 

The  virtual  image  is  erect  with  the  Dutch,  but  inverted  with  the  Keplerian 
microscope. 

§  695.  The  Dutch  compound  microscope.  —  So  far  as  known  at 
present  the  first  compound  microscope  invented  was  composed  of 
two  lenses,  a  convex  lens  for  the  objective  and  a  concave  lens  for  the 


430  HISTORY  OF  LENSES  AND  MICROSCOPES          [Cn.  XII 

ocular  (fig.  248).  The  convex  lens  is  placed  to  give  a  real  image  of 
the  object,  that  is,  the  object  is  outside  the  principal  focus  of  the 
objective  (fig.  248).  But  before  the  real  image  is  formed  a  concave 
lens  (the  ocular)  is  placed  in  the  path  of  the  beam.  This  makes 
the  rays  less  convergent  and  therefore  acts  as  an  amplifier  to  in- 
crease the  size  of  the  real  image  of  the  objective.  The  eye  is  placed 
close  to  the  ocular  and  focuses  the  real  image  on  the  retina.  This 
retinal  image  is  inverted  (fig.  5-6)  and  therefore  when  pro- 
jected out  into  space  it  seems  erect  (fig.  248),  as  with  the  simple 
microscope  (fig.  182). 

From  the  testimony  of  eye-witnesses  this  form  of  compound  micro- 
scope was  devised  by  a  spectacle  maker  in  Middleburg,  Holland,  about 
the  year  1590,  the  name  of  the  inventor  being  Zacharias  Jansen. 
(See  Borellus.) 

Very  early  the  two  lenses  were  put  into  tubes  and  made  capable 
of  being  brought  together  or  separated,  depending  upon  the  distance 
of  the  object  to  be  examined.  The  nearer  the  object  the  farther  apart 
must  be  the  ocular  and  objective.  There  still  remains  in  the  ordinary 
opera  glass  the  original  Dutch  telescope.  If  one  has  an  opera  glass 
it  is  easily  demonstrated  that  it  can  be  used  as  a  microscope  by  un- 
screwing the  ocular  so  that  it  may  be  separated  a  considerable  distance 
from  the  objective.  If  now  the  objective  is  held  within  10  to  20 
centimeters  of  an  object  and  the  ocular  moved  back  and  forth  along 
the  axis,  the  place  will  be  soon  found  where  the  image  is  distinct  and 
it  will  be  seen  much  enlarged. 

The  name  telescope  was  given  sometime  before  1618,  and  the  desig- 
nation microscope  in  1825  (§  2a).  As  every  one  who  used  the  instru- 
ment found  that  it  could  be  used  as  a  microscope  or  as  a  telescope 
it  soon  came  to  be  called  a  telescope-microscope,  or  a  microscope- 
telescope. 

§  696.  The  Keplerian  compound  microscope.  —  When  the  Dutch 
telescope  came  to  the  attention  of  the  astronomer  and  optician, 
Kepler,  he  very  quickly  saw  that  the  same  effect  could  be  brought 
about  by  using  a  convex  ocular  as  well  as  a  convex  objective,  but  that 
the  image  would  be  inverted,  the  objective  serving  to  produce  an 
enlarged  real  image  and  the  ocular  to  magnify  that  image. 


Cn.Xiri          HISTORY  OF  LENSES  AND  MICROSCOPES  431 

The  demonstration  of  the  principles  on  which  such  a  microscope  or 
telescope  could  be  constructed  is  to  be  found  in  the  Dioptrica  of  Kepler, 
Proposition  LXXXVI.  The  proposition  is:  With  two  convex  lenses 
to  show  objects  larger  and  inverted. 

In  Prop.  LXXXIX,  it  is  stated  that  with  three  convex  lenses 
can  be  shown  objects  enlarged  and  erect.  This  is  the  principle 
of  the  terrestrial  or  erecting  telescope. 

Kepler  first  showed  the  real  action  of  the  eye  as  an  optical  instru- 
ment, and  that  the  retinal  image  must  be  inverted,  and  that  unless 
inverted,  objects  would  appear  wrong  side  up.  Now  we  know  that  is 
true,  for  it  is  an  easy  demonstration  to  show,  as  did  Scheiner  in  1619- 
1625,  that  the  retinal  image  is  actually  inverted  in  the  eye  of  an 
animal  or  man. 

As  Kepler  showed  the  actual  dioptrics  of  the  eye,  he  was  the 
first  to  explain  the  real  action  of  spectacles  in  correcting  the 
defects  of  long  sight  and  short  sight,  viz.  to  aid  the  refracting 
surfaces  of  the  eye  to  make  a  sharp  image  of  the  object  upon  the 
retina. 

While  Kepler  gave  the  optical  demonstration  for  a  microscope  or 
telescope  with  convex  lenses,  he,  so  far  as  known,  did  not  actually 
construct  such  a  microscope  or  telescope.  Christopher  Scheiner, 
while  he  lacked  the  original  genius  of  Kepler  for  discovering  and  ex- 
pounding principles,  had  greater  mechanical  ability.  He  actually 
constructed  the  Keplerian  telescope  and  microscope  and  used  them 
both  for  observation  and  for  projecting  real  images.  On  page  130  of 
the  Rosa  Ursinae  (1626-1630)  occurs  this  remarkable  passage:  "  In 
the  same  way  [i.e.  by  two  convex  lenses]  was  produced  that  wonderful 
microscope  by  which  a  fly  was  made  as  large  as  an  elephant  and  a  flea 
to  the  size  of  a  camel." 

§  697.  Binocular  microscopes.  —  From  the  first  invention  of  the 
telescope-microscope  there  was  dissatisfaction  that  it  was  for  but  one 
eye,  and  before  1610  there  were  made  those  for  both  eyes  by  putting 
two  equal  instruments  side  by  side  the  right  distance  apart  for  the 
eyes  of  the  observer.  That  arrangement  of  the  Dutch  telescope  still 
holds  in  opera  glasses. 

One  of  the  first  examples  shown  in  pictured  form  is  that  of  the 


432  HISTORY  OF  LENSES  AND  MICROSCOPES          [Cn.  XII 

Cherubin  d'Orleans  in  1677  (fig.  250).  This,  as  seen  from  the  picture, 
is  a  binocular  Keplerian  microscope,  or  rather  two  of  them,  as  both 
objectives  and  oculars  are  of  convex  lenses.  The  objectives  needing 
to  be  close  together  makes  a  divergence  of  the  tubes  necessary  to  get 
the  right  pupillary  distance  for  the  oculars.  In  general  this  form  of 
binocular  has  been  recently  revived  for  dissection,  only  in  the  modern 
form  achromatic  objectives  are  used  and  Huygenian  oculars,  and  by 
means  of  prisms  the  image  is  made  erect. 

Only  rather  large  objects  can  be  studied  with  such  binoculars,  and 
the  effort  to  divide  the  light  from  a  single  objective  reached  success 
only  as  late  as  1851,  when  it  was  worked  out  by  J.  L.  Riddell  of  New 
Orleans.  His  description  and  a  figure  were  published  in  the  Quarterly 
Journal  of  Microscopical  Science  in  1854.  From  that  time  on  success- 


FIG.  250.    BINOCULAR  MICROSCOPE  OF  CHERUBIN  D'ORLEANS. 

ful  binocular  microscopes  have  been  made.  The  one  of  Wenham 
(fig.  52)  in  England  (1860)  enjoyed  the  greatest  favor.  Tolles  in 
1864-1865  produced  his  binocular  eye-piece,  and  Nachet,  in  France, 
and  Zeiss,  in  Germany,  produced  binocular  instruments,  but  there  were 
defects  inherent  in  the  construction  of  all  forms,  especially  the  defect 
that  they  could  not  be  used  very  satisfactorily  with  high  powers,  and 
they  were  expensive.  Finally,  in  1902,  Mr.  F.  E.  Ives  figured  and 
described  a  form  of  binocular  suitable  for  all  powers  including  the 
nighest  oil  immersions  (§  142,  150).  Several  recent  models  have  been 
produced  in  which  the  principles  he  enunciated  so  clearly  have  been 
incorporated  (fig.  53,  54,  55). 

In  the  first  binoculars  of  the  Dutch  form  the  tubes  were  parallel, 
as  with  the  opera  glass,  but  in  many  of  the  later  forms  (fig.  52,  250) 
and  many  others  the  tubes  were  made  divergent.  With  others,  as 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES  433 

the  binoculars  of  Nachet  (1853)  and  Harting  (1858),  the  tubes  are 
parallel  (§  144). 

§  698.  Microscopes  for  two  or  more  observers.  —  The  projection 
microscope  with  its  real  images  on  a  screen  has  been  commended  from 
the  first  invention  of  projection  apparatus  because  many  can  see  the 
image  at  the  same  time,  and  the  teacher  or  exhibitor  can  be  sure  that 
the  observers  are  seeing  the  special  things  he  wishes  to  show.  But 
in  looking  into  the  microscope  in  the  ordinary  way  only  one  person 
can  look  at  a  time,  even  with  the  ordinary  binocular.  Therefore  there 
arose  the  effort  to  divide  the  light  from  the  object  so  that  two  or  more 
could  see  the  same  image  at  the  same  time.  The  use  of  prisms  for 
dividing  the  light  in  the  binocular  gave  the  hint,  and  in  1853  Nachet 
constructed  a  microscope  for  two  observers,  and  another  for  three 
observers  (see  figures  of  these  in  Harting  and  in  Robin's  work  on  the 
microscope,  also  in  the  original  paper).  Harting,  1858,  also  produced 
a  microscope  for  two  observers.  For  this  the  tubes  were  parallel.  By 
putting  them  closer  together  they  served  for  a  binocular  for  one  person. 

Finally,  in  his  enthusiasm  for  demonstration,  he  constructed  a  mi- 
croscope in  which  the  beam  was  divided  among  four  diverging  tubes 
so  thjkt  four  persons  could  see  the  same  specimen  at  once. 

Within  recent  years  the  demand  for  a  way  by  which  two  observers 
could  look  at  once  has  given  rise  to  two  very  practical  double  oculars 
which  are  far  enough  apart  so  that  two  can  look  into  the  oculars  con- 
veniently. One  was  devised  (1910)  by  Dr.  Edinger  of  Frankfurt  and 
produced  by  Ernst  Leitz  in  Germany,  and  the  other  in  1916,  by  the 
Spencer  Lens  Company  of  Buffalo,  New  York.  In  both  these  double 
oculars  there  is  an  adjustable  pointer  so  that  the  exact  structure  which 
is  to  be  studied  can  be  indicated;  then  both  teacher  and  student  can 
be  sure  that  they  are  talking  about  the  same  thing. 

§  699.  Oculars.  —  As  shown  above  the  first  oculars  were  of  single 
lenses,  —  for  the  Dutch  telescope-microscope  a  concave  lens,  and  for 
the  Keplerian  microscope  a  convex  lens. 

For  the  Keplerian  microscope,  which  soon  became  the  only  one 
used  for  microscopic  work,  all  sorts  of  experiments  were  tried  both 
for  oculars  and  for  objectives.  Finally,  about  1660,  Huygens,  the 
great  Dutch  astronomer  and  physicist,  designed  for  the  telescope 


434 


HISTORY  OF  LENSES  AND  MICROSCOPES          [Cn.  XII 


the  ocular  (fig.  23-24)  which  now  bears  his  name.    It  was  soon  adopted 
for  the  microscope  and  is  to  this  day  the  most  used  of  any. 

The  Ramsden  ocular  was  devised  by  J.  Ramsden  (1782)  for  the 
telescope  and  like  the  Huygenian  was  adapted  to  the  microscope.  It 
has  been  used  especially  for  the  ocular  micrometer  (fig.  22  A,  93). 

The  Compensation  oculars  were  invented  by  Abbe  (1885-1886) 
to  go  with  the  apochromatic  objectives  and  to  correct  the  residual 
defects  in  the  objectives  (fig.  22  B,  174-175). 

§  700.  Mirrors  and  condensers  for  illuminating  objects.  —  The 
first  objects  looked  at  through  the  microscope,  whether  simple  or 

compound,  were  opaque  and 
must  be  illuminated  by  light 
falling  upon  their  surface. 
For  this  were  used  condensing 
lenses,  plane  and  concave  mir- 
rors. The  origin  of  the  mirror 
is  prehistoric.  The  first  were 
of  polished  metal  and  of  dark 
minerals.  Those  with  a  metal 
backing  have  been  known  only 
since  about  the  i2th  or  i3th 
century,  and  those  with  silver 
only  since  about  100  years  ago. 
It  is  not  to  be  forgotten  that 
still  water  and  other  smooth 
objects  in  nature  serve  as  mir- 
rors, and  have  always  existed. 
In  Descartes'  picture  of  the 
Dutch  compound  microscope 
(fig.  251)  there  is  a  parabolic 
mirror  for  lighting  the  object  if 
opaque,  and  a  condensing  lens 
for  transparent  objects.  Des- 
cartes also  gives  a  picture  of  a 
simple  microscope  with  a  similar  concave  mirror  for  illuminating  the 
opaque  object  (fig.  252).  In  1668  Hooke  speaks  of  looking-glasses 


FIG.  251.  DESCARTES'  DUTCH  COM- 
POUND MICROSCOPE  WITH  A  PARABOLIC 
MIRROR  AND  A  CONDENSING  LENS. 

abc,  def,     Concave  ocular  (amplifier). 

5  T  Stand  and  circle  holding  the  mi- 
croscope and  pointing  it  toward  the  sun  or 
other  light  source. 

N  O  P    Convex  objective. 

C  C  Parabolic  mirror  for  illuminating 
opaque  objects. 

i  i  Condenser  for  illuminating  trans- 
parent objects. 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES 


435 


for  illuminating  transparent  objects  for  projection.  The  first  pictures 
of  compound  microscopes  with  the  mirror,  as  at  present  under  the 
stage,  are  by  Hertzel  (1712)  and  Marshall  (1718). 

A  condenser  of  a  single  lens  or  of  a  combination  of  lenses  for  trans- 
parent objects  dates  from  the  earliest  use  of  the  compound  microscope, 
as  shown  by  Descartes'  figure.  Its  importance 
for  adequate  lighting  has  never  been  lost  sight 
of,  as  indicated  by  Brewster  (§  looa)  and  by 
Nelson  (see  in  collateral  reading);  and  never 
so  thoroughly  appreciated  as  at  the  present 
day.  The  form  most  common  on  microscopes 
is  the  uncorrected  one  of  Abbe  which  was  first 
described  in  the  Archiv  fur  Mikr.  Anat.  Vol. 
9,  1873,  p.  469. 

§  701.  Achromatization.  —  As  pointed  out 
in  §  463-464,  white  light,  being  composed  of 
different  wave  lengths  (fig.  144-146),  must  be 
differently  refracted  when  passed  through  a 
prism  or  lens.  To  the  normal  human  eye  the 
different  waves  when  separated  or  dispersed 
out  into  groups  appear  of  different  colors. 
Although  the  nomenclature  used  by  Newton 
was  somewhat  different  from  that  now  used, 
he  supposed  that  the  refraction  of  the  differ- 
ent waves  was  in  exact  accordance  with  their 
wave  lengths,  as  is  the  case  with  a  diffraction 
grating,  and  hence  there  could  be  no  achro- 
matization  of  dioptric  instruments,  for  when 
the  dispersion  was  overcome  the  refraction 
must  also  be  eliminated.  The  mistaken  belief 
that  the  human  eye  was  achromatic,  however, 
kept  alive  the  hope  of  producing  achromatic 
microscopes  and  telescopes.  Experiments  on  a  large  number  of 
transparent  substances  showed  that  while  all  dispersed  the  light,  the 
dispersion  was  not  the  same  in  all,  some  affecting  one  group  out  of 
proportion  to  another.  This  irregularity  gave  the  clue  to  the  way  to 


FIG.  252.    DESCARTES' 
SIMPLE  MICROSCOPE. 

1 1  Rays  of  light  pass- 
ing to  the  reflector. 

C  The  parabolic  re- 
flector for  illuminating 
the  opaque  object. 

A  The  plano-convex 
lens  serving  as  a  mag- 
nifier. 

GE  Pin  for  holding 
the  opaque  object. 

H  Crystalline  lens  of 
the  eye. 


436  HISTORY  OF  LENSES  AND  MICROSCOPES  [Cn.  XII 

accomplish  achromatism,  for  if  two  or  more  transparent  bodies  could 
be  combined  and  neutralize  their  dispersive  effect  without  overcoming 
the  mean  refraction  it  would  be  possible  to  make  achromatic  combina- 
tions. This  is  shown  by  the  course  of  the  beam  of  white  light  travers- 
ing the  two  prisms  (fig.  172).  The  first  to  accomplish  the  feat  in  a 
way  to  make  achromatic  telescopes  possible  was  John  Dollond  (1757). 
Naturally  the  telescope  took  the  lead  in  the  improvement,  as  it  at  that 
time  was  by  far  the  most  important  optical  instrument.  Furthermore, 
the  lenses  were  relatively  large;  for  in  the  differentiation  of  the  tele- 
scope and  microscope  the  objective  of  the  telescope  became  pro- 
gressively larger  and  that  for  the  microscope  progressively  smaller. 
The  smaller  the  lenses  the  more  perfect  must  be  the  grinding  and 
polishing,  for  slight  imperfections  in  their  small  area  introduce 
obscurations  which  in  the  larger  surface  of  the  telescope  lenses  would 
be  negligible  (§  476,  fig.  180).  But  the  microscope  makers  undertook 
the  task  in  several  different  countries,  —  England,  France,  Russia, 
Holland,  Germany,  and  Italy  —  and  from  1759  to  1824  were  tireless 
in  their  efforts.  Finally  Selligue  laid  before  the  French  Academy  the 
result  of  his  efforts  with  the  help  of  the  practical  opticians,  Vincent 
and  Charles  Chevalier.  From  that  time  on  achromatic  objectives 
became  more  and  more  common  for  microscopes,  although  from  their 
small  aperture  they  were  not  liked  by  some  workers  so  well  as  the 
more  brilliant,  uncorrected  lenses. 

In  our  own  country,  Charles  A.  Spencer  took  the  lead  in  trying  to 
overcome  the  lack  of  brilliancy  in  achromatic  objectives.  He  too, 
early  realized  and  grasped  the  importance  of  aperture  for  the  micro- 
scopic objective.  He  realized  also  that  for  the  balancing  of  the  dis- 
persions and  refractions  to  make  true  achromatic  combinations,  it  was 
necessary  to  have  materials  for  lenses  with  special  properties.  He 
worked  in  two  directions.  One  was  the  use  of  the  natural  mineral 
fluorite  whose  properties  had  been  pointed  out  by  Brewster  (§  465%) 
and  the  other  was  the  production  of  new  forms  of  glass  with  specially 
desired  optical  qualities. 

It  fills  one  with  admiration  to  think  of  this  genius  with  small  means 
working  alone  in  his  cramped  quarters  trying  to  make  new  forms  of 
glass,  which  with  the  old  forms  and  with  natural  minerals  would  enable 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES  437 

him  to  produce  the  objectives  of  his  dream  with  large  aperture  and 
perfect  color  and  spherical  correction.  While  his  success,  and  that  of 
his  pupil  Tolles,  were  certainly  great  in  producing  the  highest  type  of 
objective  for  the  telescope  and  microscope  with  the  materials  already 
to  be  had,  his  glass  making  did  not  bring  him  all  that  he  wanted.  It 
was  reserved  for  the  optical  works  of  Zeiss  and  the  genius  of  Abbe, 
with  the  help  of  the  practical  glass  maker  Schott,  and  the  liberality 
of  the  German  government  to  finally  overcome  the  difficulties  in  mak- 
ing new  forms  of  glass  with  specially  desired  qualities  of  dispersion 
and  refraction;  and  even  then  it  was  necessary  to  go  back  to  the 
natural  mineral  fluorite  to  make  possible  the  apochromatic  objec- 
tives. Those  interested  are  recommended  to  read  the  work  of  Hove- 
stadt  on  the  new  Jena  glass. 

§  702.  Immersion  objectives.  —  In  the  development  of  any  art 
the  science  needed  almost  always  lags  behind,  and  is  developed  in 
most  cases  to  explain  what  has  already  been  discovered  by  the  hard 
and  roundabout  method  of  "  trial  and  error."  This  was  the  case 
with  immersion  objectives.  Amici  in  Italy  and  David  Brewster  in 
Great  Britain  were  busy  in  trying  to  improve  microscope  objectives 
by  any  feasible  method.  They  used  all  sorts  of  liquids  for  immersion. 
Water  was  one  of  the  most  successful  and  still  holds  its  own. 

§  703.  Homogeneous  immersion  objectives.  —  The  advantage  of 
the  immersion  principle  gradually  became  understood  to  be  the 
possibility  of  increasing  the  aperture  under  which  the  object  could  be 
viewed.  The  final  step  by  which  the  aperture  could  be  pushed  to  the 
limit  of  human  skill  in  figuring  the  lenses  came  when  Mr.  Tolles  (1871- 
1874)  showed  in  the  clearest  manner  the  possibility  of  making  such 
objectives  and  increasing  the  aperture  by  means  of  homogeneous 
contact  between  the  condenser  and  the  slide  or  object  and  between 
the  object  or  cover-glass  and  the  front  lens  of  the  objective.  The  mat- 
ter is  well  stated  by  Hon.  J.  D.  Cox  in  his  presidential  address  before 
the  American  Microscopical  Society  for  1884  (pp.  5-39),  and  in  Mr. 
MayalFs  Cantor  Lectures  on  the  History  of  the  Microscope  (1885). 
On  p.  96  Mayall  says:  "  If  priority  of  publication  of  the  formula  on 
which  homogeneous  immersion  objectives  could  be  produced  carries 
with  it  the  title  of  inventor,  then  Mr.  R.  B.  Tolles  stands  alone  as 


438  HISTORY  OF  LENSES  AND  MICROSCOPES  [Cn.  XII 

inventor;  but  he  not  only  published  the  formula,  he  -constructed 
objectives  on  it."  The  formula  was  submitted  with  the  objective 
in  1874.  The  homogeneous  immersion  objectives  of  Zeiss  came  out 
in  1878. 

Many  substances  have  been  tried  for  the  homogeneous  fluid. 
Thickened  cedar-wood  oil  has  proved  most  satisfactory.  Mr.  Tolles 
used  Canada  balsam;  if  one  gets  out  of  cedar- wood  oil  and  has  Canada 
balsam  of  moderate  thickness,  good  results  can  be  obtained  by  using 
the  balsam  as  an  immersion  liquid. 

§  704.  Projection  microscope.  —  The  production  of  real  images 
by  means  of  a  naked  aperture  and  by  means  of  a  lens  were  the  begin- 
nings of  the  magic  lantern,  the  photographic  camera,  the  projection 
microscope,  and  the  drawing  camera. 

As  shown  elsewhere  (Optic  Projection,  p.  673),  the  production  of 
real  images  in  dark  places  by  means  of  an  aperture  or  hole  in  the  wall 
is  a  purely  natural  phenomenon.  The  systematic  utilization  of  this 
phenomenon  by  man  had  its  beginnings  in  the  sixteenth  and  seven- 
teenth centuries.  The  first  certain  statement  of  the  use  of  a  lens  in 
the  aperture  to  make  the  picture  clear  and  vivid  occurs  in  the  work 
of  Daniel  Barbaro  on  perspective  (§  705a). 

From  this  time  on  a  lens  is  always  used  for  projection.  At  first 
the  images  were  smaller  than  the  object,  as  naturally  only  the 
brightly  lighted  objects  in  the  exterior  world  were  projected,  but  as 
artificial  and  natural  light  were  used  to  illuminate  smaller  and  smaller 
objects,  many  of  which  were  transparent,  and  the  projection  lenses 
were  made  of  shorter  focus,  the  images  became  larger  than  the  object. 
Finally  (1665),  when  the  apparatus  became  small,  and  only  the  object 
and  lens  and  light  were  enclosed  and  the  image  was  on  a  screen  out- 
side, the  magnifying  action  seemed  like  that  of  a  microscope,  and 
Milliet  de  Chales,  in  speaking  of  the  magic  lantern  of  Walgensten, 
says  (Vol.  II,  p.  667) :  "  In  this  machine  you  have  a  kind  of  micro- 
scope," and  Zahn,  p.  255,  in  discussing  the  magic  lantern,  says:  "  It  is 
a  kind  of  a  microscope."  Both  authors  point  out  the  great  advantage 
this  kind  of  a  microscope  has  over  the  ordinary  one  in  that  many  per- 
sons can  see  the  image  at  the  same  time.  Kepler  (1611)  showed  that 
the  Dutch  telescope-microscope  could  be  used  for  projecting  images 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES  439 

and  also  his  own  combination  of  convex  lenses.  Scheiner  (1626- 
1630)  used  them  for  projecting  images  of  the  sun  so  that  he  could 
draw  the  spots.  See  also  Hooke,  Trans.  Roy.  Soc.,  1668,  p.  741. 

Naturally,  with  the  perfecting  of  objectives  (1824  and  onward), 
and  the  finding  of  more  powerful  artificial  lights  (lime  light,  1824, 
electric  light,  especially  since  1880),  the  projection  microscope  is  coming 
to  be  used  more  and  more. 

§  705.  Drawing  magnified  images.  —  The  first  drawings  made  by 
the  aid  of  the  microscope  were  free-hand.  Examples  of  the  drawings 
may  be  seen  in  the  work  of  Borellus,  and  in  facsimiles  shown  in  the 
Journal  of  the  Royal  Microscopical  Society,  1915,  pp.  317-340.  The 
desire  for  accuracy  and  ease  in  tracing  outlines  of  microscopic  images 
comparable  with  those  so  easily  attained  with  the  real  images  of 
the  projection  microscope  led  to  the  invention  of  the  camera  lucida, 
by  which  the  microscopic  field  and  the  drawing  field,  pencil,  etc.,  can 
be  superposed.  The  first  one  invented  is  still  used.  It  is  the  Woilas- 
ton  form  (fig.  99),  and  was  described  by  Wollaston  in  Nicholson's 
Journal,  1807,  pp.  1-5.  The  other  form  shown  in  fig.  100  was  de- 
scribed in  principle  by  G.  Burch,  Jour.  Quek  Micr.  Club,  1878, 
p.  47;  and  by  Dippel  in  the  Bot.  Centrlbl.  1882,  pp.  242-3. 

Drawing  with  the  projection  apparatus  has  been  practised  from  its 
first  invention.  Indeed,  in  all  those  who  described  such  apparatus, 
the  great  help  that  was  to  be  gained  in  drawing  was  emphasized.  Both 
•eyes  can  be  used,  and  perfect  freedom  of  the  artist  is  enjoyed,  which 
is  in  marked  contrast  with  camera  lucida  drawing.  For  the  early 
appreciation  of  projection  apparatus  and  the  camera  obscura  for 
drawing  see:  Barbaro,  1568  (§  705a);  Kepler,  1611  (§  705b);  Scheiner, 
1626-1630;  Robert  Hooke,  1668;  Baker,  1742  (§705c);  Adams, 
1746;  Goring  and  Pritchard,  1837;  Chevalier,  1839. 

705a.  Daniel  Barbaro.  —  In  his  work,  La  pratica  della  perspettiva,  Venice, 
1568,  Ch.  V,  p.  192,  Barbaro  says:  "Take  an  old  man's  glass,  convex  on 
both  sides,  not  concave  like  the  glasses  of  youths  of  short  sight,  fix  the  convex 
glass  in  a  hole,  close  all  the  windows  so  that  no  light  may  enter  except  through  the 
lens.  Now  take  a  sheet  of  white  paper  and  bring  it  toward  the  lens  until  all  outside 
the  house  is  clearly  seen.  When  the  proper  position  is  found  you  will  see  the  images 
on  the  paper  as  they  are,  and  the  gradations  in  colors,  shadows,  movements,  clouds, 
the  rippling  of  waters,  birds  flying,  and  everything  that  can  be  seen.  For  this 
experiment  the  sun  must  be  clear  and  bright,  for  the  sunlight  has  great  power  in 


440  HISTORY  OF  LENSES  AND  MICROSCOPES          [Cn.  XII 

bringing  out  the  images.  You  can  draw  on  the  paper  with  a  pencil  all  the  perspective, 
and  the  shading  and  coloring  according  to  nature." 

705b.  Johannes  Kepler.  —  In  Reliquiae  Wottonianae,  edited  by  Izaak 
Walton,  London,  1672  pp.  298-300.  In  a  letter  to  his  kinsman,  Francis  Bacon: 
"I  have  your  Lordship's  letters  dated  the  2oth  of  October  (1620).  I  lay  a  night 
at  Lintz  .  .  .  there  I  found  Kepler,  a  man  famous  in  the  sciences,  as  your  Lord- 
ship knows,  to  whom  I  purpose  to  convey  from  hence  one  of  your  books  [Novum 
Organum],  that  he  may  see  we  have  some  of  our  own  that  can  honor  our  king  as 
well  as  he  has  done  with  his  Harmonica. 

In  this  man's  study  I  was  much  taken  with  a  draught  of  a  landskip  on  a  piece 
of  paper,  me  thought  masterly  done;  whereof  enquiring  of  the  author,  he  bewrayed 
with  a  smile,  it  was  himself;  adding  he  had  done  it,  non  tanquam  pictor,  sed  tan- 
quam  mathematicus  [not  as  an  artist  but  as  a  mathematician].  This  set  me  on 
fire:  At  last  he  told  me  how.  He  hath  a  little  black  tent  (of  which  stuff  it  is  not 
much  importing)  which  he  can  suddenly  set  up  where  he  will  in  a  field;  and  it  is 
convertible  (like  a  windmill)  to  all  quarters  at  pleasure,  capable  of  not  much  more 
than  one  man,  as  I  conceive,  and  perhaps  at  no  great  ease;  exactly  close  and  dark, 
save  at  one  hole,  about  an  inch  and  a  half  in  diameter,  to  which  he  applies  a  long 
perspective  trunk  [Dutch  Telescope]  with  the  convex  glass  fitted  to  the  said  hole 
and  the  concave  taken  out  at  the  other  end,  which  extendeth  to  about  the  middle 
of  this  erected  tent;  through  which  the  visible  radiations  of  all  the  objects  without 
are  intromitted,  falling  upon  a  paper  which  is  accommodated  to  receive  them;  and 
so  he  traceth  them  with  his  pen  in  their  natural  appearance,  turning  his  little  tent 
around  by  degrees  till  he  hath  designed  the  whole  aspect  of  the  field.  This  I  have 
described  to  your  Lordship  because  I  think  there  might  be  good  use  made  of  it 
for  chorography;  for  otherwise  to  make  landskips  by  it  were  illiberal,  though 
surely  no  painter  could  do  them  so  precisely." 

§  706c.  Henry  Baker.  —  The  Microscope  Made  Easy,  1742.  On  page  25  occurs 
this:  "Such  too  as  have  no  skill  in  drawing  may,  by  this  contrivance,  [projection 
microscope],  easily  sketch  out  the  exact  figure  of  an  object  they  have  a  mind  to 
preserve  a  picture  of;  since  they  need  only  fasten  a  paper  upon  a  screen  and  trace 
it  out  thereon  either  with  a  pen  or  pencil  as  it  appears  before  them." 

§  705d.  Dark -field  microscopy.  —  The  ancient  opticians  appreciated  as  keenly 
as  we  at  the  present  day,  that  the  principle  of  contrast  was  of  the  highest  import- 
ance in  rendering  objects  visible;  but  before  this  could  be  applied  to  the  micro- 
scope, the  instrument  itself  must  be  invented  (1590-1611);  and  then  must  follow 
the  perfecting  of  all  the  optical  parts  and  the  invention  of  a  reflecting  condenser 
(1850-1856),  and  the  working  out  of  the  immersion  principle  to  its  culmination  in 
the  homogeneous  immersion  objectives  of  Tolles  (1871-1874).  For  artificial  light 
the  arc  lamp  of  Davy  (1800)  and  the  Mazda  lamps  of  our  own  time  were  needed, 
and  finally  the  Daylight  Glass  to  protect  the  eyes  and  clarify  the  image. 

In  glancing  backward  over  the  long  road  which  has  been  traversed  in  arriving  at 
the  present  stage  with  optical  instruments,  there  are  two  causes  for  astonishment: 
First,  that  mankind  was  so  late  in  discovering  the  laws  of  refraction,  and  then  their 
application  for  the  production  of  lenses  and  their  combination  into  optical  instru- 
ments; and  secondly,  the  almost  fabulous  progress  that  has  taken  place  since  the 
first  possibilities  of  lenses  were  discovered  some  six  hundred  and  fifty  years  ago, 
and  especially  during  the  last  three  hundred  and  fifty  years  since  the  combination 
of  lenses  to  make  the  compound  microscope  and  the  telescope  was  found  out. 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES  441 

COLLATERAL  READING  FOR  CHAPTER  XII 
(For  the  full  titles,  when  not  given,  see  the  general  bibliography.) 

ABBE,  E.  —  Substage  condenser,  Apochromatic  objectives,  etc.,  Hovestadt  and 
Zeiss  catalogues. 

ADAMS,  G.  —  Micrographia,  1720-1773.     Essays,  1750-1795. 

AIRY,  G.  B.  —  On  a  peculiar  defect  in  the  eye  and  a  mode  of  correcting  it.  Cam- 
bridge Philos.  Trans.,  Vol.  II,  1827,  pp.  267-271.  Here  is  discussed  astigma- 
tism and  its  correction  by  cylindrical  glasses. 

ALHAZEN.  — The  Opticae  Thesaurus,  translated  by  Risner,  1572. 

BACON,  ROGER.  —  Opus  Majus,   1266-1267.     Combach's  edition,   1614.     On  p. 
159  occurs  the  description  of  a  convex  glass  to  aid  old  men  in  reading. 
Bridges  edition,  1897-1900.    Vol.  II,  p.  157,  spectacle  for  old  men. 
Opus  tertium,opus  minus.    Brewer,  in  Compendium  studii  philosophiae,  1859. 
Part  of  the  Opus  tertium,  including  a  fragment  now  printed  for  the  first 
time.    Edited  by  A.  G.  Little,  1912.    On  p.  40  is  repeated  the  statement  about 
the  convex  lens  for  old  men,  and  much  other  optics. 

BAKER,  H.  —  Microscopes,  1742,  p.  25. 

BARBARO,  DANIEL.  —  La  pratica  della  perspettiva,  Venice,  1568. 

BAUSCH  &  LOMB  OPTICAL  COMPANY.  —  Lenses,  their  History,  theory  and  manu- 
facture. Published  in  honor  of  the  ninth  annual  convention  of  the  Ameri- 
can Association  of  Opticians.  Rochester,  1906.  47  pages,  many  figures. 

BOCK,  E.  —  Die  Brille  (spectacles)  und  ihre  Geschichte,  1903. 

BORELLUS,  PETRUS.  —  De  vero  telescopii  inventore,  1655.  Dutch  compound 
microscope  by  eye-witnesses. 

BREWSTER,  SIR  DAVID.  —  The  Edinburgh  encyclopaedia,  1832;  treatise  on  the 
microscope,  1837. 

CARPENTER-DALLINGER.  —  Revelations  of  the  Microscope,  1901. 

Cox,  HON.  J.  D.  —  Robert  B.  Tolles  and  the  angular  aperture  question.  Deals 
with  the  origin  of  the  homogeneous  immersion  objective  also.  Transactions 
of  the  Amer.  Micr.  Soc.,  1884,  pp.  5-39. 

CHEVALIER,  CH.  —  Des  microscopes,  1839. 

DESCARTES,  R.  —  Dioptrique.  1637. 

DOLLOND,  J.  —  Philos.  Trans.  Roy.  Soc.,  London,  1758,  pp.  733-743.  An  account 
of  some  experiments  concerning  the  different  refrangibility  of  light.  Every 
one  interested  in  optical  instruments  ought  to  read  this  paper. 

EDINGER.  —  See  tor  description  of  the  double  ocular,  Jour.  Roy.  Micr.  Soc.,  1911, 
p.  252. 

GAGE,  S.  H.  &  H.  P.  —  Optic  Projection,  1914. 

FaAVos,  K \au5ios.  —  Hepl  XP«as  r&v  kv  avdpuirov  (runart  nopiuv  (Galenus, 
Claudius.  —  De  usu  partium  corporis  humani,  131-201  A.D.). 

GORING  &  PRITCHARD.  —  Micrographia,  1837. 

HARTING,  P.  —  Het  Mikroskoop,  1858;   Das  Mikroskop,  2d  ed.,  1866. 


442  HISTORY  OF  LENSES  AND  MICROSCOPES          [CH.  XII 

HOOKE,  R.  —  Philos.  Trans.  Roy.  Soc.,  1668,  p.  741. 

HOVESTADT,  H.  —  Jena  Glass,  1902. 

HUYGENS.  —  For  HUYGENS'S  ocular  see  Nelson,  Jour.  Roy.  Micr.  Soc.,  1900, 
pp.  162-169. 

JOURNAL  OF  THE  ROYAL  MICROSCOPICAL  SOCIETY.  —  In  nearly  every  number  is  the 
announcement  of  some  new  thing  pertaining  to  the  microscope.  For  the  special 
purposes  of  this  chapter  attention  is  called  especially  to  the  volume  of  1886,  pp. 
849-856,  for  the  apochromatic  objectives;  to  that  of  1889,  in  which,  on  pp.  574- 
598  is  a  translation  of  a  paper  by  the  eminent  Italian  physicist,  G.  Govi,  on  the 
invention  of  the  compound  microscope.  He  contends  that  it  was  Galileo  who 
first  found  that  the  Dutch  telescope  could  also  be  used  as  a  microscope.  This 
paper  may  well  be  read  in  connection  with  the  book  of  Borellus. 

In  1891,  pp.  90-105,  Mr.  Nelson  deals  with  the  substage  condenser,  and  in 
1900,  pp.  162-169,  with  the  history  of  the  Huygenian  ocular.  In  1902, 
pp.  20-23  Mr.  Nelson  gives  a  bibliography  of  works  (dated  not  later  than 
1700)  dealing  with  the  microscope  and  other  optical  matters.  In  1914  Dr. 
Jentzsch,  pp.  1-16,  and  Conrad  Beck,  pp.  17-23,  205-210,  deal  with  binocular 
microscopes,  past  and  present. 

In  1915  Charles  Singer,  pp.  317-340,  deals  especially  with  early  drawings 
made  by  the  aid  of  the  microscope;  and  in  1916,  Mr.  Heron- Allen  and  Ch.  F. 
Rousselet  give  a  summary  of  the  progress  of  knowledge  of  vision  and  the  mi- 
croscope from  1673-1848. 

KEPLER,  J.  —  Opera  omnia,  Vol.  n.  See  in  Paralipomena,  Caput  V,  for  the 
action  of  the  eye  and  of  spectacles,  especially  pages  255-262.  And  in  the 
Dioptrica,  pp.  529-556,  for  the  eyes  and  for  combinations  of  lenses. 

MAYALL,  J.  —  History  of  the  microscope,  1885. 

MILLIET  DE  CHALES.  —  Mundus  mathematicus,  1674.  Figure  of  Walgensten's 
magic  lantern,  Vol.  II,  p.  666. 

MOLYNEUX,  WM.  —  Dioptrica  nova,  1692.    Excellent  on  history. 

NACHET.  —  Sur  un  nouveau  microscope  approprie  aux  besoins  des  demonstrations 
anatomique,  et  permettant  a  plusieurs  personnes  d'observer  ensemble.  Compte 
rendu  des  stances  de  la  Societe  de  Biologie,  Oct.  1853.  pp.  141-145.  In  this 
article  is  described  and  illustrated  forms  of  microscopes  with  two  tubes  for 
two  observers,  and  with  three  tubes  for  three  observers.  See  also  Harting, 
Vol.  Ill,  pp.  243-248.  See  also  plates  I-II  in  the  Dutch  edition  of  1858. 

NEWTON,  SIR  ISAAC.  —  Optics,  1704. 

PANSIER,  P.  —  Histoire  des  lunettes  (spectacles),  1901. 

PETRI,  R.  J.  —  Das  Mikroskop  (History). 

PRIESTLEY,  J.  —  History.    Vision,  light,  and  colors.    1772. 

PTOLEM^EUS,  C.  —  Optics  (70-147  A.D.). 

POGGENDORFF,  J.  C.  —  Geschichte  der  Physik,  1879.    History. 

RAMSDEN,  J.  —  Philos.  Trans.  Roy.  Soc.,  1782.    A  description  of  a  new  construe- 


CH.  XII]          HISTORY  OF  LENSES  AND  MICROSCOPES  443 

tion  of  eye-glass  (oculars)  for  such  telescopes  as  may  be  applied  to  mathe- 
matical instruments.  (Pp.  94-98,  i  plate.  In  Vol.  LXXIII,  1783.) 

RISNER,  J.  —  See  Alhazen. 

SCHEINER,  C.  —  Rosa  Ursinae,  1826-1830.  Action  of  the  eye,  p.  108;  The  tele- 
scope and  microscope  and  projection  therewith,  p.  130. 

SPENCER  &  TOLLES.  —  See  Krauss,  Trans.  Amer.  Micr.  Soc.,  1901,  pp.  19-29. 
History  of  their  contributions  to  microscopy. 

TOLLES- SPENCER.  —  See  Cox,  Trans.  Amer.  Micr.  Soc.,  1884,  pp.  5-39;  Krauss, 
Trans.  Amer.  Micr.  Soc.,  1901,  pp.  19-29;  Mayall,  Hist.  Micr.,  pp.  95-96. 

WALGENSTEN'S  magic  lantern.     Milliet  de  Chales,  q.v. 

YOUNG,  THOMAS. — On  the  mechanism  of  the  eye,  1800,  in  Philos.  Trans.  Roy. 
Soc.  London,  1801,  pp.  23-88.  On  pp.  39-40  he  describes  astigmatism,  and 
shows  that  it  can  be  corrected  by  tilting  the  spectacles.  See  Airy. 

ZAHN,  J.  —  Oculus  artificialis  teledioptricus,  etc.,  1702.  Many  figures  of  optic 
apparatus  at  that  time. 

History  of  Dark-field  Microscopy. 

CARPENTER,  WM.  B.  —  The  Microscope  and  its  revelations,  1856-1881. 
EDMUNDS,  J.  —  On  a  new  immersion  paraboloid.     Mon.   Micr.  Jour.,  Vol.   18, 

1877,  pp.  78-85. 
GAGE,  S.  H.  —  Modern  dark-field  microscopy  and  the  history  of  its  development. 

Trans.  Amer.  Micr.  Soc.,  Vol.  39,  1920,  pp.  95-141. 
HALL,  J.  C.  —  Quart.  Jour.  Micr.  Sci.,  Vol.  IV,  1856,  pp.  205-208.     Figures  the 

"spotted  lens." 

LISTER,  JOSEPH  JACKSON.  —  Trans.  Roy.  Soc.,  Vol.  120  (1830),  pp.  187-200. 
QUEKETT,  JOHN.  —  Practical  treatise  on  the  use  of  the  microscope.     Editions  of 

1848,  1852,  1855. 

READE,  REV.  J.  B.  —  See  pp.  227-231  of  Goring  &  Pritchard. 
SHADBOLDT,  G.  —  Trans.  Micr.  Soc.  London,  Vol.  Ill  (185^),  pp.  132,  154. 
SIEDENTOPF,  H.  —  Vorgesichte  der  Spiegelkondensoren,  Zeit.  wiss.,  Mikr.,  Vol. 

XXIV  (1907),  pp.  382-395- 
WENHAM,  F.  H.  —  Reflecting  condensers,  Trans.  Micr.  Soc.  London,  Vol.  Ill,  1850, 

pp.  83-90.     Quart.  Jour.  Micr.  Sci.,  Vol.  II,  1854,  pp.  145-158.    Trans.  Micr. 

Soc.  London,  in  Vol.  IV,  1856,  Quart.  Jour.  Micr.  Sci.,  pp.  55-60. 


REDUCING  GLASS 
Convex  /^tT^  Convex 
Lens 
Ocular 
Objective" 


Lens 
Ocular 
Objective 


Simple 


Microscope 


Compound 


Microscope 


Compare  with  the  Title  Page  and  with  Fig.  4- 


BIBLIOGRAPHY 

IN  the  following  list  of  books  and  periodicals  are  mentioned  those  which 
have  furnished  most  helpful  information.  Those  which  are  still  in  print 
usually  have  the  price  given. 

For  new  information  the  reader  is  advised  to  consult  the  Journal  of  the 
Royal  Microscopical  Society;  the  Index  Medicus  and  the  Wistar  Institute 
Journals,  —  American  Journal  of  Anatomy,  Anatomical  Record,  Journal  of 
Morphology,  Comparative  Neurology  and  Journal  of  Experimental  Zoology. 
For  information  concerning  the  development  of  the  science  consult  the  Index 
Catalogue  of  the  Surgeon  General's  Office,  the  Catalogue  of  Scientific  Papers 
published  by  the  Royal  Society  of  London,  the  larger  works  on  the  micro- 
scope, and  the  microscopical  journals,  as  these  furnish  a  good  record. 

ADAMS,  GEORGE,  1720-1773.  —  Micrographia  illustrata;  or,  the  microscope 
explained,  in  several  new  inventions;  likewise  a  natural  history  of  aerial, 
terrestrial,  and  aquatic  animals,  etc.,  considered  as  microscopic  objects, 
lix  +  325  pp.  72  plates.  Published  for  the  author,  London,  1771. 

ADAMS,  GEORGE,  1750-1795.  —  Essays  on  the  microscope,  containing  a  descrip- 
tion of  the  most  improved  microscopes,  a  history  of  insects,  their  transforma- 
tions, peculiar  habits,  and  ceconomy,  with  a  catalogue  of  interesting  objects, 
xxiii  +  724  pp.  31  plates.  Published  for  the  author,  London,  1787. 

ALHAZEN.  —  Opticae  thesaurus  Alhazeni  Arabis,  libri  septem,  nunc  primum  editi, 
ejusdem  liber  de  crepusculis  et  nubium  ascensionibus,  item  Vitellionis  Thu- 
ringopoloni,  libri  X.  omnes  instaurati,  figuris  illustrati  et  aucti,  adjectis  etiam 
in  Alhazenum  commentariis.  A  Frederico  Risnero.  Folio,  many  figures, 
Basileae,  per  Episcopios.  1572. 

BACON,  ROGER.  —  Opus  Majus,  edited  with  introduction  and  analytical  table  by 
John  Henry  Bridges.  2  vols.  and  supplementary  vol.  Vol.  I,  clxxxvii  + 
440  pp.  23  fig.  Vol.  II,  568  pp.  187  fig.  Supplement,  xv  +  187  pp.  Williams 
&  Norgate,  London,  1897-1900.  315.  6d.  For  modern  optics  the  part  desig- 
nated De  Scientia  Perspectiva  is  most  important.  For  use  of  convex  lenses 
to  aid  the  sight  of  old  men,  see  vol.  ii,  p.  157,  and  for  burning  flasks,  p.  471. 

BACON,  ROGER.  —  Essays  contributed  by  various  writers  on  the  occasion  of  the 
commemoration  of  the  seventh  centenary  of  his  birth.  Collected  and  edited 
by  A.  G.  Little.  426  pp.  Clarendon  Press,  Oxford,  England,  1914.  Price, 
$5.25.  Biography  of  Bacon  and  essays  upon  his  work  in  various  fields.  List 
of  Bacon's  writings. 

445 


446  BIBLIOGRAPHY 

BAKER,  HENRY,  F.  R.  S.  —  Of  Microscopes  and  Observations  made  thereby. 
2  vols.  New  edition.  Vol.  I,  The  Microscope  made  easy.  Projection  micro- 
scope. Vol.  II,  Employment  for  the  Microscope.  London,  1785. 

BARBARO,  DANIEL.  —  La  pratica  della  perspettiva  di  Monsignor  Daniel  Barbaro, 
eletto  patriarca  d'Aquileia.  Opera  molto  utile  a  pittori,  a  scultori  &  ad  archi- 
tetti.  Con  privilegio.  208  pp.  Many  figures.  In  Venetia,  appresso  Camillo 
&  Rutilio  Borgominieri  fratelli,  al  segno  di  S.  Giorgio  MDLXVIII  (1568). 
First  known  user  of  a  lens  in  the  camera.  Cap.  V,  p.  192. 

BAUSCH,  E.  —  Manipulation  of  the  Microscope.  A  manual  for  the  work  table 
and  a  text-book  for  beginners  in  the  use  of  the  microscope.  200  pp.  Illust. 
New  edition.  Rochester,  N.Y.,  1906.  Price  $1.00. 

BEALE,  L.  S.  —  How  to  Work  with  the  Microscope.  5th  edition,  1880,  518  pages, 
99  plates  with  500  figures.  Harrison,  London.  Cost  215. 

BECK,  CONRAD.  —  The  Theory  of  the  Microscope.  Cantor  Lectures  delivered 
before  the  Royal  Society  of  Arts,  Nov.-Dec.,  1907.  59  pp.  Illust.  London, 
1908. 

BECK,  CONRAD,  and  ANDREWS,  HERBERT.  —  Photographic  Lenses.  7th  edition, 
completely  revised.  287  pp.,  163  fig.,  44  plates.  R.  &  J.  Beck,  Limited. 
68  Cornhill,  London,  England.  Price  i  shilling.  Full  discussion  of  modern 
objectives  for  photography  and  for  projection. 

BOCK,  DR.  EMIL.  —  Die  Brille  und  ihre  Geschichte.  62  pages,  frontispiece,  and 
32  text  figures.  Wien,  1903.  Verlag  von  Josef  Safar.  Price  4.20  marks. 

BORELLUS,  PETRUS.  —  De  vero  Telescopii  inventore,  cum  brevi  omnium  Con- 
spiciliorum  historia.  Ubi  de  eorum  confectione,  ac  usu,  seu  de  efTectibus 
agitur,  novaque  qusedam  circa  ca  proponuntur.  Accessit  etiam  centuria  obser- 
vationum  microcospicarum.  Authore  Petro  Borello,  regis  christianissimi 
consiliario,  et  medico  ordinario.  Hagae-Comitum,  ex  typographia  Adriani 
Vlacq,  MDCLV  (1655).  Important  for  the  history  of  optic  instruments. 

BOYER,  CHARLES  S.  —  The  Diatomaceae  of  Philadelphia  and  Vicinity.  Quarto, 
143  pages,  40  plates  (700  drawings  by  the  author  at  a  scale  of  800  diameters). 
Philadelphia,  1916.  Press  of  J.  B.  Lippincott  Company,  East  Washington 
Square.  Cost  $5. 

BREWSTER,  SIR  DAVID.  —  A  Treatise  on  the  Mikroscope.  From  the  7th  edition 
of  the  Encyclopaedia  Britannica,  with  additions.  Illust.  1837. 

BREWSTER,  SIR  DAVID.  —  The  Edinburgh  Encyclopaedia.  Optics,  Vol.  14.  Joseph 
and  Edward  Parker,  Philadelphia,  1832.  On  page  764,  2d  column,  near  middle, 
is  described  the  use  of  the  amalgam  on  the  back  of  looking-glasses  as  a  screen. 
I  have  tried  this  and  found  it  wonderfully  efficient. 

BURNETT,  SAMUEL  HOWARD.  —  The  Clinical  Pathology  of  the  Blood  of  Domesti- 
cated Animals.  156  pp.  Ithaca,  1908.  24  figures,  4  colored  plates. 

CARPENTER-DALLINGER.  —  The  Microscope  and  its  Revelations,  by  the  late  William 
B.  Carpenter.'  8th  edition,  in  which  the  ist  seven  and  the  23d  chapters  have 
been  entirely  rewritten,  and  the  text  throughout  reconstructed,  enlarged,  and 


BIBLIOGRAPHY  447 

revised  by  the  Rev.  W.  H.  Dallinger.  22  plates  and  nearly  900  wood  engrav- 
ings. 1181  pp.  London  and  Philadelphia,  1901.  P.  Blakiston's  Son  &  Co. 
Price  $8.00. 

CHAMBERLAIN,  C.  J.  —  Methods  in  Plant  Histology.  3d  editiDn  314  pages,  107 
figures.  The  University  of  Chicago  Press,  1916.  Cost  $2.25. 

CHAMOT,  £MILE  MONNIN.  —  Elementary  Chemical  Microscopy.  410  pages,  i 
plate,  139  text  figures.  John  Wiley  &  Sons,  N.Y.,  1915.  Price  $3.00. 

CHEVALIER,  CHARLES.  —  Des  Microscopes  et  de  leur  usage.    Illust.    Paris,  1839. 

CLARK,  C.  H.  —  Practical  methods  in  microscopy.    2d  ed.    Illust.    Boston,  1896. 

COMSTOCK,  ANNA  BOTSFORD.  —  Handbook  of  Nature  Study.  900  pages,  1000 
illustrations.  Comstock  Publishing  Co.  Ithaca,  N.Y.,  6th  edition,  1916. 
Price  $3.25  +  40  cents  postage. 

COMSTOCK,  JOHN  HENRY.  —  A  Manual  for  the  Study  of  Insects.  701  pages. 
798  figures,  colored  frontispiece.  Comstock  Publishing  Company,  Ithaca, 
N.Y.  i$th  edition,  1917.  Price  $3.75  +  32  cents  postage. 

CZAPSKI,  S.  —  Grundziige  de*  Theorie  der  optischen  Instrumente  nach  Abbe. 
2  AufL,  unter  Mitwirkung  des  Verfassers  und  mit  Beitragen  von  M.  v.  Rohr. 
490  pp.  Illust.  Leipzig,  1904. 

DANIELL,  A.  —  A  Text-book  of  the  Principles  of  Physics.  New  edition,  1911. 
The  MacMillan  Co.,  London  and  New  York.  Cost  $4.00. 

DESCARTES  (Lat.  Cartesius)  RENE.  —  (Euvres,  Publiees  par  C.  Adam  et  P. 
Tannery  sous  les  auspices  ministere  de  1'instruction  publique,  Vols.  i-xii. 
Dioptrique,  Vol.  6,  pp.  87-228,  73  fig.  Leopold  Cerf,  12  Rue  Sainte  Anne, 
Paris,  1902. 

DESCARTES  (Lat.  Cartesius),  RENE.  —  (Euvres;  publiees  par  V.  Cousin.  Paris, 
1824-26.  ii  vols.  Dioptrique,  Vol.  5.  pp.  1-153,  S  plates,  including 
66  fig. 

DIPPEL,  L.  —  Das  Mikroskop  und  seine  Anwendung.    Illust.    Braunschweig,  1898. 

EHRLICH.  —  Enzyklopadie  der  Mikroskopischen  Technik.  Herausgegeben  von : 
Ehrlich,  Krause,  Moose,  Rosin  und  Weigert.  2d  edition,  2  Vol.,  800  +  680 
pages,  56  +  111  figures.  Urban  &  Schwarzenberg,  Berlin  and  Vienna,  1910. 
Cost  27.50  marks. 

GAGE,  S.  H.  and  H.  P.  —  Optic  Projection.  Principles,  installation  and  use  of 
the  magic  lantern,  projection  microscope,  reflecting  lantern  and  moving  pic- 
ture machine.  731  pages,  413  figures.  Comstock  Publishing  Co.  Ithaca, 
N.Y.  1914.  Price  $3.00. 

GORING  and  PRITCHARD.  —  Micrographia,  containing  practical  essays  on  reflect- 
ing, solar,  oxy-hydrogen  gas  microscopes,  micrometers,  eye-pieces,  etc.,  etc, 
231  pp.  Many  figures  in  the  text,  one  plate.  Whittaker  &  Co.,  Ave-Maria 
Lane,  London,  England,  1837. 

Govi,  GILBERTO.  —  Galileo,  the  inventor  of  the  compound  microscope,  —  Journal 
of  the  Royal  Microscopical  Society,  1889,  pp.  574-598.  Discussion  of  the 
earliest  discoveries  and  inventions  in  optics.  The  compound  microscope  here 


448  BIBLIOGRAPHY 

referred  to  as  the  invention  of  Galileo  is  the  Dutch  telescope  used  as  a  micro- 
scope, i.e.  an  instrument  like  the  ordinary  opera  glass  with  a  longer  tube  for 
the  convex  objective  and  concave  ocular. 

GUYER,  MICHAEL  F.  —  Animal  Micrology.  Practical  exercises  in  Zoological 
micro-technique.  289  pages,  74  figures.  The  university  of  Chicago  Press, 
1917.  Price  $2.00. 

HARDESTY,  IRVING.  —  A  Laboratory  Guide  for  Histology,  with  a  chapter  on 
laboratory  drawing,  by  A.  W.  Lee.  193  pages,  30  figures.  P.  Blakiston's 
Son  &  Co.  Philadelphia,  1908.  Cost  $1.50. 

HARTING,  P.  —  De  Nieuwste  Verbeteringen  van  het  Mikroskoop  en  Zijn  Gebruik, 
Sedert  1850.  176  pages,  two  triple  plates.  Te  Tiel.  bij  H.  C.  A.  Campagne, 
1858. 

HARTING,  P.  —  Das  Mikroskop.  Theorie,  Gebrauch,  Geschichte  und  gegenwartiger 
Zustand  desselben.  Deutsche  Originalausgabe,  von  Verfasser  revidirt  und 
vervollstandigt.  Herausgegeben  von  Dr.  Fr.  Wilh.  Theile.  In  drei  Banden. 
Bd.  I,  Theorie;  Bd.  II,  Gebrauch;  Bd.  Ill,  Geschichte.  Bd.  I,  pp.  346,  134 
text  figures,  i  plate.  Bd.  II,  pp.  310;  104  text  figures.  Bd.  Ill,  pp.  452, 
231  text  figures.  Druck  und  Verlag  von  Friedrich  Vieweg  und  Sohn,  Braun- 
schweig, Zweite  wesentlich  verbesserte  und  vermehrte  Auflage,  1866.  Price 
about  $3.50. 

HOGG,  J.  —  The  Microscope,  its  History,  Construction  and  Application.  i5th  ed. 
704  pp.  900  Illust.  Rutledge,  London  and  New  York,  1898.  Much  atten- 
tion paid  to  the  polariscope.  Cost  IQS.  6d. 

HOOKE,  ROBERT. — Animadversions  on  the  Machina  Ccelestis  of  Hevelius.  p.  8. 
Published  in  1674.  It  is  in  this  place  that  Hooke  states  that  for  two  points 
to  be  seen  as  two  the  visual  angle  must  be  one  minute. 

HOVESTADT,  .  H.  —  Translated  by  J.  D.  and  A.  Everett.  Jena  Glass  and  its  scien- 
tific and  industrial  applications.  London,  MacMillan  &  Co.  Limited. 
N.Y.  The  MacMillan  Company.  1902.  419  pages,  29  figures.  Cost  $4.50. 

HOWELL,  WILLIAM  H.  —  A  Text-book  of  Physiology  for  Medical  Students  and 
Physicians.  6th  edition,  1020  pages,  306  figures.  W.  B.  Saunders  Co.,  Phila- 
delphia, Pa.  1916.  Price  $4.00. 

INDEX  Catalogue  of  the  Library  of  the  Surgeon  General's  Office  of  the  United 
States  Army.  Government  Printing  Office,  Washington,  D.C.  First  series, 
vols.  i-xvi,  1880-1895.  Second  series,  vols.  i-xxi,  1896-1916.  Book  and  peri- 
odical literature;  subjects  and  authors  in  one  continuous  alphabetical  list. 
Full  lists  of  current  literature. 

INDEX  MEDICUS.  —  Carnegie  Institute,  Washington,  D.C.     $8.00  per  year. 

JOURNAL  OF  THE  FRANKLIN  INSTITUTE,  devoted  to  science  and  the  mechanic 
arts.  1826  +  Philadelphia,  Pa.  Annual  subscription,  $5.00. 

JOURNAL  OF  THE  ROYAL  MICROSCOPICAL  SOCIETY.  1878  +.  Published  by  the 
Society  at  20  Hanover  Square,  London  W.,  England.  6  numbers  per 
year;  subscription  price,  37  shillings  6d. 


BIBLIOGRAPHY  449 

JOURNAL  OF  THE  ROYAL  SOCIETY  OF  ARTS.  London,  England.  The  65th  vol- 
ume of  the  JOURNAL  is  now  being  published  (1917). 

KEPLER,  JOHANNES.  —  Opera  Omnia,  Vol.  II.  Ad  Vitellionem  Paralipomena. 
(De  modo  visionis  et  humorum  oculi  usu.)  1604.  pp.  226-269.  n  fig. 
Correct  dioptrics  of  the  eye  here  given,  and  also  the  explanation  of  the 
effect  of  convex  and  concave  spectacles.  Dioptrica.  Demonstratio  eorum 
quae  visui  et  visibilibus  propter  conspicilla  non  ita  pridem  inventa  accidunt. 
pp.  519-567.  35  fig.  1611.  The  amplifier,  real  images,  and  erect  images. 
The  Keplerian  microscope  (modern  microscope). 

KINGSBURY,  B.  F.  —  Laboratory  Directions  in  Histology  and  Histological  Tech- 
nique, pp.  1-60  and  1-95.  The  MacMillan  Co.,  N.Y.  1916.  Price  $1.25. 

LANGERON,  M.  —  Precis  de  Microscopie;  technique,  experimentation  —  diag- 
nostic. 821  pages,  292  figures  in  the  text.  Masson  et  Cie.,  Paris.  2d  edition, 
1916.  Price  12  francs. 

LEE,  A.  B.  —  The  Microtomist's  Vade-mecum.  A  hand-book  of  the  methods  of 
microscopic  anatomy.  7th  ed.  526  pp.  1913.  P.  Blakiston's  Son  &  Co., 
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INDEX 


Aberration,  chromatic,  288 

correction  of,  285,  289,  294 

cover-glass  and  fig.  of,  78,  285-286 

spherical,  284-285,  288 
Absolute  alcohol,  348 

index  of  refraction,  273 
Absorption  spectra,  251,  259,  264 
Achromatic  condenser,  57-59 

objective,  21,  291 
Achromatism,  290,  435 
Achromatization,  290,  435 
Actinic  focus,  222 
Adjustable  objective,  22,  77,  229 

in  photo-micrography,  229 
Adjustment  of  analyzer,  267-269 

coarse  or  rapid;  fine,  27,  97 

of  objective  for  cover-glass,  77,  229 
Aerial  image,  31,  33 
Air  bubbles,   80,  112 
Albumen  fixative,  348 
Alcohol  absolute,  348 

ethyl,  348-349 

denatured,  349 

methyl,  349 

mixtures,  how  to  make,  347-348 

picric,  363 
Alcoholic  dye,  393 
Amici  prism,  247 
Amplifier,  139 

for  projection,  201 
Amplification  of  microscope,  131 
Analyzer,  264-266 
Angle  of  aperture,  294 

critical,  275 

visual  and  fig.,  6,  127,  128-129 
Angstrom  and  Stokes  law,  252-253 
Angular  aperture,  294-296 
Anisotropic  objects,  268 
Apertometer,  298 


Aperture,  angular  and  numerical  deter- 
mination of,  297 

numerical,  294-299 

and  diaphragm,  62-63 

and  opacities,  303-304 

table  of,  296 
Aplanatic  objectives,  21 
Apochromatic  condenser,  58 

objectives,  291,  437 

Apparatus  and  material  for  chapters, 
36,  77,   107,  127,  160,  206,  246, 
270,  3!2,  368 
Apparent  size  of  objects,  9 
Appearances,   interpretation,    107,    126 
Arc  lamp  and  fig.,  72 
Arrangement  of  minute  objects,  336 

of  serial  sections,  404 
Artificial  illumination,  48-52 
Autochrome  photographic  plates,    244- 

245 
Avoidance   of   inversion    in    drawings, 

172-176 
vibrations    in   photography,    217- 

218 
Axial  light,  65 

experiments  with,  65 
Axis,  optic,  282-284 

of  projectioti  microscope,  200-201 
secondary,  n,  284 


Back  combination,  20 

Bacteria,  334-335 

Bacterial  cultures,  photographing,  213 

Balsam,  339,  350 

acid,  351 

alkaline  or  neutral,  350 

bottle,  328-329 

filtering,  350 

mounting  in,  329-330 


453 


454 


INDEX 


Balsam,  natural,  350 

neutral  or  alkaline,  350-351 

removal  from  lenses,  93 

removal  from  slides,  315 

xylene,  350 

Base  of  microscope  (fig.),  27 
Basket  for  slides  (fig.),  385 
Bench,  optical,  221 
Bent-neck  bottle  or  vial,  331 
Bibliography,  35,   76,   126,    159,    205, 
245,  269,  311,  367,  422-423,  440, 

443 

Binoculars,  82-91,  431-432 
figures  of,  84-85,  87,  432 
parallel  or  converging-tubes,  86 

Black  aniline  for  tables,  365-366 

Blocks  for  shellvials,  332 

Blood,  absorption  spectrum  of,  259 

Blotting  paper  for  models,  413-422 

Board,  reagent,  332,  345 

Borax  carmine,  351 

Bottle  'for  balsam,  glycerin,  or  shellac, 
328-329 

Box,  imbedding  and  fig.,  378 

washing  for  tissues  (fig.),  369 

Brownian  movement  or  pedesis,  118 

Burning  point  or  focus,  284 


Cabinet  for  microscopic  preparations, 

340-345 

Calipers,  micrometer,  318-319 
Camera,  206 

drawing,  176 

photographic,  206* 

photo-micrographic,  217-218 

testing,  212 

vertical,  207-218 

vibrations,  avoidance  of,  217-218 
Camera  lucida,  136-137,  161 

Abbe  (fig.),  163-170 

Wollaston's  fig.  of,  136,  162 
Canada  balsam,  329,  350-351 

mounting  in,   329-330 

removal  from  lenses,  93 

removal  from  slides,  315 
Carbol- turpentine,  352 
Carbol-xylene,  352 


Carbon-^nonoxide  haemoglobin  spectum> 

261 
Card,  black-pinhole  (fig.),  6,  9,  10 

catalogue,  337 

centering,  326 
Care,  of  eyes,  94-96 

microscope,  mechanical  parts,  92 

optical  parts,  92-94 

of  negatives,  214 

of  oil  immersions,  82 

of  water  immersion  objectives,  80 
Carmine,  borax,  351 

muci-,  351 

spectrum  of,  262 
Castor-xylene  clarifier,  391 
Cataloguing  formula,  338-339 

preparations,  337 
Cedar- wood  oil,  352 

clearing  with,  352 

for  oil  immersion  objectives,  438* 
Cells  for  mounting,  324 

staining  isolated,  332 
Cement,  shellac,  364 
Cementing  collodion,  353 
Center,  optic  or  optical,  u,  282 
Centering  condensers,  65 

projection  apparatus,  201 

source  of  illumination,  65 
Centering-card,  326 
Central  light,  46 
Chamber,  moist,  333 
Chloral  haematoxylin,  359 
Chloroform,  352 
Chromatic  aberration,  288    ' 

correction,  290,  435 
Circle,  Ramsden's  circle,  34,  76 
Clamp  for  microtome,  378 
Clarifier,  castor-xylene,  353 
Class  demonstrations,  193,  195 
Cleaning,  back  lens  of  objective,  94 

homogeneous  immersion  objectives, 
82 

mixture  for  glass,  320 

optical  parts,  92 

slides  and  cover-glasses,  315-317 

water  immersion  objectives,  80 
Clearer,  330,  352 
Clearing  mixtures,  352 


INDEX 


455 


Cloudiness  of  objective  and  ocular,  109 

removal  of  cloudiness,  93,  109 
Coarse  adjustment  of  microscope,  27, 97 
Cobweb  micrometer,  148 
Collection  of  microscopic  material,  334 
Collodion,  353 

castor-xylene  method  with,  391 

coating  glass  rod,  116 

cementing,  353 

clarifying  of,  391 

fastening  sections  to  slide,  386 

handling  sections  with  paper,  390 

hardening,  388 

method,  387-392 

method  with  paraffin,  391-392 

transferring  sections  from  knife  to 

slide,  390 

Collodionizing  sections,  353 
Color  photography,  244-245 
Color-correct  photography,  234-240 
Color  images,  80 

law  of,  253 

production  of  by  polariscope,  267 

screens,  234-240 

spectral,  248 

Combinations  in  objectives,  20-21 
Comparing  two  microscopic  fields,  122 
Comparison  ocular  (fig.),  123 

prism,  249 

spectrum,  253 
Compensation  ocular  (fig.),  24,  292-294 

434 

Complementary  spectra,  253 

Concave  lenses,  281,  427 
mirror,  use  of,  54 

Condensers,  57-76,  297,  434 
Abbe,  59 

achromatic,  57,  59 
apochromatic,  58 
centering,  59-60 

homogeneous  immersion,  64,  297 
image  of  scale  for  micrometry,  155 
in  drawing  for  models,  419-420 
lighting  the  field  with,  63 
mirror  with,  63 
non-achromatic,  58-59 
numerical  aperture  of,  60 
photo-micrography  with,  228-231 


source  of  light  with,  60-6 1 

standard  size,  106 

substage,  57 
Condensing  lens  for  projection,  size  of, 

233,  420 

Congo  glycerin,  354 
Congo  red,  354 
Construction  of  images,  12 

real  and  virtual  images,  13-14 
Continuous  spectrum,  248 
Contour,  double,  116 
Converging  lens,  281,  427 
Convex  lens,  281 
Correction,  chromatic  or  color,  288-294 

of  the  aberration  of  lenses,  284-294 

cover-glass,  285 

over  and  under  correction,  285-288 
Cotton,  collodion,  353 

soluble,  353 

Counterstaining,  395-396 
Cover-glass  or  covering  glass,  313 

aberration,  (fig.)  78,  285-286 

adjustment,  directions,  78-79,  229 

adjustment    for    in   photo-microg- 
raphy, 229 

adjustment  and  tube  length,  286- 
288 

anchoring,  327 

cleaning,  316-318 

correction,  286-288 

effect  on  rays  from  object,  40-43, 
286 

focusing  with  and  without,  42-43 

for  unadjustable  objectives,   285- 
287 

gauges  or  measures  (fig.),  318-320 

measuring  the  thickness  of,  43-45. 
318-320 

sealing,  326-327 

thickness,  318-319 

tube-length    and    cover-thickness, 
286-288 

variation  in  thickness,  319 

wiping,  317-318 

working  distance  with  and  fig., 40-43 
Critical  angle,  64,  275-276 
Crystals  for  pedesis,  119 
Currents,  diffusion,  avoidance,  117,  396 


456 


INDEX 


Cutting    sections,    free-hand,    paraffin, 

collodion,  374~375>  3&7 
Cyco  photographic  paper,  178 
Cylindrical  spectacles,  428 

D 

Dark-ground  condenser  or  illuminator, 

71-73 

illumination,  67-76,  120,  297 
Dark   room   for   drawing  and  photog- 
raphy, 217,  242 
Daylight,  45 

artificial  with  curve,  49-53,  203-205 

glass,  50-53 

lantern  with,  203-205 

table  with,  203-205 
Decalcifier,  354 
Dehydration,  395 
Demonstration,  class,  193-205 

lantern  and  table,  203-205 

projection  microscope,  197-203 
Denatured  alcohol,  349 
Deparaffining,  384 
Designation  of  oculars,  23-26 

of  wave  lengths  of  light,  256 
Determination  of  field,  29-30 

magnification,  136,  307 

working  distance,  41 
Developing  and  light  for,  241-242,  243 
Diamond  for  writing,  401 
Diaphragms  and  their  use,  47,  48,  62, 

181 

Diatomes,  335-336 
Dichromate  cleaning  mixture,  320 
Diffraction  of  light,  280,  300-302 
Diffuser,  ground-glass  for,  50-53,  181 
Diffusion  currents,  avoidance,  396 
Direct  light,  45 

vision  spectroscope,  249 
Disc,  Ramsden's,  34 
Dispersion  of  light,  280 
Dissecting  microscope,  88-90 

spectacles,  87 
Dissociating  liquids,  354 
Dissociator,  332,  354 

Formaldehyde,  354 

Muller's  fluid,  354 

nitric  acid,  354 


Distance,  standard  for  microscopy,  139 

working,  39-40 
Distinct  vision,  near  point,  for  adults, 

130 

Distinctness  of  outline,  114 
Distortion  in  drawing,  162 
Diverging  lens,  281 
Dividers,  measuring  spread  of,  134 
Double-objective  binocular,  83-88 

ocular,  433 
Double  vision,  134 
Doubly  contoured,  116 

refracting,  268 
Draw-tube,  27,  37 
Drawing,  160-193 

board  for  camera  lucida,  166,  169 

camera  lucida,  161-170 

distortion,  avoidance  of,  162 

erect  images  in,  172-176 

free-hand,  161 

inversion  in,  172-176 

magnification  of,  309 

microscopic,  162-174 

models,  416-420 

photographic  camera  for,  161,  173, 
176-178 

projection  apparatus  for,  161,  183- 
188 

room  for,  242 

scale  and  enlargement,  170-172 

table  and  shelf  for  and  fig.,  179, 186 

with  low  and  high  powers,  186,  187 

with   the  simple   microscope,  188, 

190 
Drawings,  lettering,  190-191 

on  back  of  photographic  prints,  178 

on  blue  prints,  178 

on  a  vertical  or  horizontal  drawing 
board,  185 

reduction  for  publication,  190 
Dry  mounting,  323 

objectives,  and  aperture,  21,  296- 
297 

plates,  244 

Drying  rack  for  bottles  (fig.),  366 
Dust  of  living  rooms,  examination,  123- 
124 

on  objectives  and  oculars,  34,  109 


INDEX 


457 


Dutch  compound  microscope  (fig.),  429 
Dyes  in  microscopy,  392-393 


Eikonometer  (fig.),  I54~i5S 

micrometry  and  magnification  with, 

IS4-I55 

Elastic  stain,  355,  397 
Electric  arc  lamp,  417-419 

incandescent  for  projection, 41 7-420 
Electrification  of  paraffin  ribbons,  381 
Embryos,  camera  for,  210 

photographing,  209 

serial  sections  of,  402 
Enlargements  of  photographs,  232-234 

focusing  and  printing  for,  233 
Eosin,  355-356 

counterstaining  with,  395-396 
Eosin-methylene  blue,  356,  398 
Equivalent    focus,   oculars  and  objec- 
tives, 19 

Erect  images,  174-176 
Erecting  binocular,  88 
Ether-alcohol,  356 

sulphuric,  356 
Ethyl  alcohol,  348 
Examination  of  dust,  etc.,  in  rooms,  123- 

124 

Excluder  of  light  (fig.),  226-227 
Experiments,  15,  28-34,  37-40,  53-56, 
61-70,   77-82,    88-91,    109-125, 
224-231,     258-264,      267-269 

adjustable   and   immersion   objec- 
tives, 77-82 

binocular  microscopes,  88-91 

compound  microscope,  28-34 

dark-ground  or  field,  67-75 

focusing,  37-40 

interpretation  of  appearances,  109- 
125 

lighting,  53-70 

micro-polariscope,  267-269 

micro-spectroscope,    258-264 

mounting,  321 

photo-micrography,  224-231 

simple  microscope,  15 
Exposure  for  photographs,    240-241 

with  color  screen,  241 


Eye  and  microscope,  5 

visual  acuity,  127 

visual  angle,  128 
Eyes,  care  of,  94-96 

muscae  volitantes  in,  122 

unlikeness  of  the  two,  91 
Eye-lens  of  the  ocular,  25,  308-309 
Eye-piece  or  ocular,  23 

demonstration,  433 

micrometer,  143,  153 

parfocal,  38-39,  309 
Eye-point,  15,  33 

demonstration  of,  33 


Farrant's  solution,  356 
Field  and  fig.  of,  29,  1 10 

camera  lucida,  161-162 

illumination  of,  48,  63,  109-110 

view  with  microscope,  29, 
Field-lens  of  ocular,  33,  308-309 

dust  on,  109 

Filar  micrometer  ocular,  147-149 
Filtering  balsam,  350 
Fine  adjustment,  27,  97-98 
Fir,  balsam  of,  350 
Fixation,  368 
Fixative,  albumen,  348 
Fixer,  368 

Flemming's  fluid,  356 
Fluid,  Muller's,  362 

Zenker's,  366 

Fluorite  lens,  n,  292,  436-437 
Focus,  n,  284 

actinic  and  visual,  222 

appearances  with  different,  121 

equivalent  of  objectives  and  ocu- 
lars, 19,  27 

depth  of  and  aperture,  303-304 

principal,  11-12,  284 
Focus,  real,  12-14 

virtual,  12 

visual  and  actinic,  222 
Focusing,  15,  36 

adjustments,  testing,  97-98 

binocular  microscope  for  the  two- 
eyes,  89 

compound  microscope,  37-38 


458 


INDEX 


Focusing,  effect  of  cover-glass  on,  42- 

43 

experiments,  36-38 

for  printing  enlargements,  233-234 

glass,  210-212 

high  objectives  and  low,  37 

in  photo-micrography,  224 

screen  for  photography,  210-211 

simple  microscope,  15,  36 

slit  of  micro-spectroscope,  254 

stand,  210 

Form  of  objects,  determination  of,  in 
Formaldehyde,  formalin,  356 

dissociator,    354 

isolation  with,  332 

percentages,  347 
Formula  for  aperture,  295 

for  cataloguing  specimens,  338-339 

for  desired  percentages,  346 
Fraunhofer  lines,  251 
Free-hand  sections,  374 
Free  working  distance,  39-40 
Front  combination  or  lens  of  objective, 

21 

Frontal  sections,  407 
Fuchsin,  acid,  364 

basic,  355 

picro,  364 
Function  of  objective,  31,  32 

of  ocular,  32 


Gauge  for  cover-glasses,  318-319 

Gauze  absorbent,  315 

Gelatin,  361 

Geometrical  construction  of  images,  12, 

281 
Glass  cleaning  mixture,  320 

ground,  31 

rod,  appearance  under  microscope, 

US 

slides  or  slips,  312-317 

varnish  for  writing  on,  401 
Glasses,  graduate,  348 

watch,  249,  259 
Glycerin,  357 

Congo,  354 

mounting  objects  in,  325 


Glycerin  jelly,  357 

for  anatomic  preparations,  358 
for  microscopic  preparations,    326- 

3a7»357 

Glycogen,  iodin  stain  for,  359,  398 

Graduate  glasses,  348 

Ground- glass  focusing  screen,  31,  210- 

211 

preparation  of,  31 
Gun  cotton,  353 

H 

Hardening  collodion,  388 

tissue,  368-369 
Hematin,  359 
Hematoxylin,  chloral,  359 

iron,  359 

staining  with,  394 
Hemoglobin  spectrum,  260 
High  School  microscope,  99 
Highly  refractive,  116 
Histology,  physiologic,  340 
History  of  lenses  and  microscopes,  424- 
442 

of  photo-micrography,  216 
Holder  for  magnifier,  17 

for  sectioning  paraffin,  378 
Homogeneous  immersion  condenser,  64, 
297 

cleaning,  82 

experiments  with,  81-82 

history  of,  437-438 

liquid,  21,  299-300,  352,  438 

numerical  aperture  of,  295-296 

objectives,  21,  295,  437 

tester,  299-300 
Hones  and  honing,  372-373 
Hood  for  front  of  objective  and  fig., 

203 
Huygenian  ocular,  25-26,  146 

for  movable  scale  micrometer,  146 

for  photo-micrography,  221 

I 
Illumination,  45-76 

air  and  oil,  112-114 
artificial,  54~75 
camera  lucida,  163-170 


INDEX 


459 


Illumination,  centering  sources  of  light, 

65 

dark -ground,  67-76,  120 

daylight,  45,  53 

entire  field,  48,  63,  109-110 

micro-polariscope,  267 

micro-spectroscope,  257 

oblique  with  air  and  oil,  112-113 

opaque  objects,  46, 434-435 

photo-micrography,  222 

Wollaston's  camera  lucida,   163 
Illuminator  or  condenser,  57 
Image,  absorption,  235 

aerial,  31,  33 

color,  80 

erect,  34,  174-176 

formed  by  lenses,  281-284 

geometrical  construction  of,  12-14 

inverted  real  of  objective,  34 

magnification  of  real  and  virtual, 
133,  140,  176 

real  fig.  of,  13-14 

refraction,  80 

retinal,  7,  34 

size  and  position,  133-139,  176 

swaying,  66 

tracing,  173,  178-181 

virtual,  8,  14,  15,  34,  139-141 
Imbedding,  377-378,  387-392 

double  in   collodion  and  paraffin, 

391-392 
Immersion,  21,  64,  437 

condenser  or  illuminator,  64,  297 

fluid  or  liquid,   21,   299-300,  352, 
437-438 

objective,  21,  47,  80,  437~438 
Incandescence  or  line  spectra,  250 
Incident  light,  45 
Index  of  refraction,  272,  295 

absolute  and  relative,  273-274 

table  of  substances,  277 
Indicator  ocular,  102,  195,  433 
Infiltration,  376,  387 

collodion,  387-388 

dish  and  oven,  377 

double  with  collodion  and  paraffin, 
391-392 

paraffin,  376-384 


Infusions  and  infusoria,  335 

infusoria    for    dark-ground  illumi- 
nation, 70 
Initial  or  independent  magnification,  19, 

305 

Ink  for  labels,  etc.,  339-340 
Interpretation  of  appearances,  107 

dark-ground   illumination    to   aid, 

120 

summary  for,  125 
Inversion  of  the  image,  124 
Inversion  in  drawing,  avoidance  of ,  172, 

176 

Invisible  and  visible  radiation,  246-247 
lodin  in  alcohol,  361 

for  removing  mercuric  chlorid,  361- 

362 

stain  for  glycogen,  359,  398 
Iris  diaphragm,  47 
Isochromatic  plates,  235 
Isolation,  330 

formaldehyde,  332 
nitric  acid,  333 
Isotropic  objects,  267 


Japanese  filter  (lens)  paper,  93 

Jar  for  slides,  360,  363,  385 

Jelly,  glycerin,  357 

Jena  glass,  437 

Jurisprudence,  rm'crometry  in,  157,   159 


Kerosene  lamp,  61 
Knife,  sharpening  of,  372 
support,  380 

L 

Labels  and  catalogues,  337 
Labeling  microscopic  preparations,  337- 
338 

photographic  negatives,  214 

serial  sections,  410 
Laboratory  compound  microscope  table. 

55,95 
desk,  96 
lockers,  345 
Lagrange  disc  or  circle,  34 


460 


INDEX 


Lamp,  alcohol  or  spirit,  382 

arc,  fig.  of,  72-74 

black  for  ingestion,  361 

electric,  62 

kerosene  and  mazda,  61,  74 
Lantern,  for  daylight  glass  and  fig.,  51 

slides,  182-183 
Lateral  swaying  of  image,  66 
Law  of  color,  253 
Lens,  10,  280-286 

aberration  of,  284-285 

converging,  281 

convex,  281 

eye,  25,  433 

field,  33 

fluorite,  292,  436-437 

focus  on  both  sides  and  fig.  12 

history  of,  424-429 

holder  and  fig.,  17 

images  formed  by  and  fig.,  280-284 

paper,  bibulous  paper,  93 

spherical,  281-283 
Lettering  oculars,  26 
Letters  or  figures,  mounted,  31 

for  drawings,  190-193 

in  stairs,  no 

white  for  black  back-ground,  192 
Light,  and  lighting,  artificial,  48-49 

artificial  daylight,  49-53,  65 

axial,  46,  65 

central,  46 

dark-ground,  67 

daylight,  45 

direct  and  central,  45-46 

direct,  45 

electric,  49,  74,  184,  204,  230-231, 
418-419 

excluder  for    camera    and    micro- 
scope (fig.),  226-227 

experiments,    54 

for  developing  photographs,   241- 
242 

for  photography  and  photo-microg- 
raphy, 209,  222,  228 

incident,  45 

micro-polariscope,  267 

micro-spectroscope,  25, 

mirror  with,  53-54 


oblique,  47 

polarized  light,  264-270 

reflected,  45,  272 

sunlight,  45 

transmitted  light,  46 

wave  length  of,  270 

with  kerosene  lamp,  54 
Line  spectrum,  250 
Lintless  towels,  315 
Liquids,  currents  in,  117 

gelatin,  361 

homogeneous,  438 
Locker,  laboratory  or  student,  345 
Longisections,  399 

M 

Magnification,  131,  136 

compensation  oculars,  306-308 

determination  of,  136 

effect  of  adjusting  objective  on,  144, 

.  I57 
eikonometer  for,  154,  155 

expressed    in    diameters   or    times 
linear,  131 

initial  or  independent,  19,  305 

of  compound  microscope,  4,  46,  132 

of  objective,  19,  305 

of  photo-micrographs,  228 

of  projection  apparatus,  170,     208, 
228 

of  real  images,  133 

of  simple  microscope,  133-140 

of  virtual  images,  135 

relation   of    object   and    principal 
focus,  140-142 

table  of,  143 

varying    with     compound    micro- 
scope, 139 

velocity  and,  117 
Magnifier,  7,  16 

tripod,  1 6 

Marker  for  preparations,  100-102 
Marking  negatives,  214 

objects,  197 

preparations,  100-101 
Masks  for  preparations,  199-200 
Mazda  gas-filled  lamp,  55,  74 
Measurer  for  cover-glasses,  318,  319 


INDEX 


461 


Measuring  thickness  of  slides  and  cov- 
ers, 319-320 

spread  of  dividers,  134 
Mechanical  parts  of  compound  micro- 
scope, 27,  99 

care  of,  92 

Mechanicabstage,  102 
Mercuric  chlorid,  362 

crystals,  361-362 
Methemoglobin  spectrum,  261 
Method,  collodion,  387 

paraffin,  375 
Methyl  alcohol,  349 
Methylated  spirits,  349 
Methylene  blue,  356 

alkaline,  362 

and  eosin,  356,  398 
Micrometer,  135,  143 

arrangement  of  ocular  and  stage, 
MS,  158 

calipers,  318 

cobweb,  148 

filar,  147-149 

filling  lines  of,  136 

net,  no,  156 

object  or  objective,  135 

ocular  and  stage,  135 

ocular  or  eye-piece,  143-1 53 

ocular,  valuation  of,  143-145 

ocular,  varying  the  valuation  of, 

144,  157 

photo-micrography  and,  228 
screw  ocular,  146 
stage  and  ocular  (fig.),  135,  136 
table  of  magnification,  143 
Micrometry,  146-159 

adjustable  objectives  in,  139,   144, 

157 

comparison  of  methods,  153-157 
compound  microscope,  150,  159 
condenser,  image  of  scale,  155 
eikonometer,  eiconometer,  149, 154- 

155 

in  jurisprudence,  157-159 
limit  of  accuracy,  158 
ocular  micrometer,  152 
remarks  on,  157-159 
simple  microscope,  149 


Micrometry,  unit  of  measure  in  (M),  150 
Micro-millimeter,  150 

micron  (/x),  mikron,  150 
Micro-photograph,     photo-micrograph, 

215 
Micro-polariscope,  264-269 

experiments  with,  267-269 
Micro-projection,  184,  189,  197 

drawing  with,  184,  189 

magnification  of,  170 

masks  for  specimens  in,  199-200 
Microscope,  4-6,  429,  430 

amplification  of,  131 

binocular,  82-91,  431-433 

care  of,  92-94 

double  objective,  83,  88 

Dutch,  429-430 

erecting,  88 

experiments  with,  88-89,  91 

field,  29-30 

fig.  of  field,  84-85,  87 

focusing,  15,  36,  37-38,  42-43,    89, 
224 

history  of,  428-439 

magnification,  131 

photo-micrography  with,  217-231 

polarizing,  264-269 

projection,  and  history,  197,438-439 

simple,  7,  133-140 

stand  for  embryos,  210 

solar,  198 

traveling,  195 
Microscope,  compound,  7,  16 

cost  of,  100 

drawing  with,  170-172 

Dutch  and  Keplerian  forms,  429- 
^430 

high-school,  99-100 

history  of,  429-439 

laboratory,  99-100 

magnification  with,  131 

mechanical  parts,  27,  99 

micrometry  with,  150-159 

optical  parts,  16-27 

quality  and  cost,  99 

testing,  97-99 

varying  magnification  of,  139,  157 

working  distance,  39 


462 


INDEX 


Microscopes  for  two  or  more  observers, 

432-433 
Microscope,  simple,  7 

drawing  with,  160-172 

experiments  with,  15 

focusing,  36 

images  with,  7,  9 

magnification  of,  133 

micrometry  with,  149 

mounting  of,  16-17 

working  distance,  39 
Microscope-telescope,  430 
Microscopic  objectives,  17 

objects,  drawing,  160,  193 

photography,  214-224 

slides  or  slips,  313 

tube-length,  27,  79,  286-287 
Microscopic  preparations,  cabinet  for, 
342 

cataloguing  and  labeling,  337-340 

mounting,  321 

trays  for,  343~345 
Micro-spectroscope,  246-264 

adjusting,  253 

Amici  prism  for,  247 

Angstrom  scale  for, -248,  255 

direct  vision,  249 

experiments  with,  258 

focusing  the  slit,  254 

in  photo-micrography,  239 

lighting,  257 

material  needed,  258 

objectives  to  use,  257 

reversal  of  colors,  248 

slit  mechanism,  253-255 
Microtomes,  370-371 

knives  for,  372,  380 
Micrum,  micron  (/*),  150 
Mikron,  micron  (/*),  150 
Milk-globules  to  overcome  pedesis  of, 

118 

Minerals,  absorption  spectra,   263 
Minute  objects,  arranging,  336 
Mirror,  434 

arrangement  for  drawing,  165-185 

central  and  oblique  light  with,  56 

concave,  use  of,  54 

dark-ground  illumination,  68,  74 


parabolic  of  Descartes,  434 

plane,  use  of,  54 
Models,  410-423 

blotting  paper  for,  413-416 

dissectable,  422 

drawings  for,  416-420 

size  of,  414-415 

thickness  of  plates  for,  414-416 

wax  for,  412-413 
Moist  chamber,  333 
Molecular  movement  or  pedesis,  118 
Monazite  sand,  spectrum  of,  263 
Mounting,  321-337 

cells,  preparation  of,  324 

media  and  preparation,  350,  356- 

357 

objects  for  polariscope,  267 
permanent,  322 
temporary,  321 
Mounting  in  balsam,  321 
dry  or  in  air,  323 
in  glycerin  and  glycerin  jelly,  326- 

327 

in  media  miscible  with  water,  325 
in  resinous  media  after  drying,  328 
in    resinous   media   by   successive 
displacements,  329-330 

Movement,  Brownian  or  molecular,  118 

Muci-carmine,  396 

Miiller's  fluid,  362 
dissociator,  354 

Muscae  volitantes,  122 

Muscular  fibers,  isolation  of,  333 

N 

Natural  balsam,  350 
Negative  oculars,  23 
Negatives,  photographic,  177 

opaquing,  182 

storing  and  labeling,  214,  232 
Net  micrometer,  no,  156 
Neutral  balsam,  350 

red,  362 
Nicol  prism,  264-265 

Nitric  acid,  362 

dissociator,  362-363 
Nomenclature  of  objectives,  19-23 
Non-achromatic  condenser,  58-59 


INDEX 


463 


Non-adjustable  objectives,  77 

thickness  of  cover-glasses  for,  286- 
287 

Normal  liquid,  363 

salt  or  saline  solution,  363 

Nose-piece,  revolving,  28-29 

Numerical  aperture  and  table,  294-297 

O 

Object,    determination    of    form,    in 

image,  relative  size  of,  138-139 
<  micrometer,  135 

mounting,  321 

printing  image  of  direct,  234 

suitable  for  photo-micrography,  223 
Objective,  19 

achromatic,  21,  291,  436 

adjustable,  22,  157,  28*5-287 

adjustment,  77,  157,  229 

aerial  image,  31,  33 

aperture,  294,  297 

aplanatic,  21 

apochromatic,  22,  291,  437 

back  combination  of,  21 

cleaning,  92-93 

cloudiness  or  dust  on,  34 

collar,  graduated,  79 

designation  or  nomenclature,  19-23 

dry,  21 

equivalent  focus  of,  19,  27 

field  of ,  29 

focusing  for  micro-spectroscope,  258 

front  combination,  21 

function  of  in  the  microscope,  31-32 

homogeneous  immersion  and  clean- 
ing, 21,  82 
experiments  with,  81-82 

immersion,  21 

initial  magnification,  19,  305 

inverted,  real  image  by,  34 

low  and  high,  20-23 

magnification  of,  19,  131,  305 

micro-polar iscope,  267 

microscopic,  19-23 

micro-spectroscope,  257 

names  of  parts,  21 

nomenclature  or  designation  of,  19- 
23 


non-adjustable,    unadjustable,    22, 

157,  285-287 
nose-piece  for,  28-29 
numbering  or  lettering,  19 
numerical  aperture,  294-297 
oil  immersion,  21,  295,  438 
pantochromatic,  22 
para-chromatic,  22 
parfocal,  38-39 
photo-micrographic,  220-222 
projection,  201 
screw-thread  for,  103-106 
semi-apochromatic,  22 
table  of  field,  29-30 
terminology  or  designation,  19-23 
unadjustable,  non-adjustable,     22, 

157,  285-287 
variable,  22 

visual  and  actinic  foci  in,  222 
water  immersion,  21,  80 
working  distance,  39-40 
Oblique  light,  47,  65-67 
Oculars,  various  forms  of,  23 

cloudiness  or  dust  on,  93,  109 
comparison  and  fig.,  123 
compensation,  24,  26,  292-293 
concave  or  amplifier,  429-434 
designation    or    nomenclature    of, 

23-26 

equivalent  focus,  27 
eye-point  of,  15-33 
field-lens,  25,  308-309 
filar  or  screw  micrometer,  147-149 
for  two  observers,  433 
function  of,  32 

Huygenian  (fig.),  6,  25-26,  146-147 
indicator  or  pointer,  102,  195 
lettering  and  numbering,  26 
magnification  of,  27,  308-309 
micrometer  and  micrometry,    143- 

153 

negative,  23 
parfocal,  38,  309 

photo-micrography,    220-222,    231 
pointer  or  indicator,  102,  195 
positive,  23 

power  or  magnification  of,  26-27 
projection,  201,  226 


464 


INDEX 


Oculars,  Ramsden's  and  fig.,  24-25 

spectroscopic,  247 

standard  sizes,  106 
Oil  and  air,  distinguishing,  113-114 

cedar- wood,  352 
Oil-globules,  study  of,  113 
Oil  immersion  objectives,  21,  295,  437 
Opacities  and  aperture,  303-304 
Opaque  objects,  lighting,  46,  434-435 
Opaquing  negatives,  182 
Opera  glasses,  430 
Optic  axis,  282-284 
Optical  bench,  221 

center,  n,  282 

parts  of  compound  microscope,  16- 
27 

section,  117 

Optics  of  the  microscope,  270-311 
Order  of  procedure  in  mounting,  323- 

329 

in  air  or  dry,  323 
in  balsam,  328-329 
in  glycerin  and  glycerin  jelly,  326- 

327 

Ordinary  ray  with  polarizer,  264 
Organs  and  tissues,  preparation  of,  368 
Orthochromatic  plates,  235 
Outlines,  distinctness  of,  114 
Oven  for  paraffin  infiltration,  377 
Over-correction,    285-288 
Oxy-hemoglobin  spectrum,  260 


Panchromatic  photographic  plates,  235 
Paper,  bibulous  (lens  paper),  93 

blotting  for  models,  413-422 

photographic  for  prints,  178,  181, 

232,  234 
Paraffin,  363,  375-386 

deparaffining,  384 

filtering,  363 

infiltrating  with,  376-377 

imbedding  in,  377 

imbedding  in  paraffin  and  collo- 
dion, 391-392 

method  for  sectioning,  375,  386 

oven  for  infiltrating,  376 

removing  from  sections,  384 


sections,  spreading,  382-384 

wax,  363 
Parfocal  objectives  and  oculars,  38-39, 

309 

Parfocalization,  38-39,  310 
Pedesis  or  Brownian  movement,  118 

overcoming,  118 

with  polariscope,  119 
Penetration  of  objectives,  302-304 
Percentage  of  liquids  and  solutions,  346 
Permanent  mounting,  322-332 
Permanganate  of  potash,  spectrum,  252,      » 

259 
Photo-engraving,  drawing  and  lettering 

for,  187-193 
Photographic  camera,  206,  210 

enlargements,  233 

magnification  rod  for,  208-210 

negatives,  177 

objectives,  178-180 

prints,  178,  181,  232,  234 
Photography,  206-245 

avoidance  of  distortion  in,  207-208 

avoidance  of  vibration,  219 

bacterial  cultures,  213 

color  correct,  234-240 

embryos,  209-212 

focusing,  210-211,  224 

for  making  figures,  176-182 

indebtedness    to    photo-microgra- 
phy, 216 

lighting  for,  209 

living  objects,  207 

objects  in  liquids,  207 

plates,    panchromatic    and    spec- 
trum, 235,  236-237 

scale   of   magnification   or    reduc- 
tion of  photographs,  208 

vertical  for  (fig.),  207,  210,  218 

scale   of   magnification    or   reduc- 
tion of  photographs,  208 

vertical  for  (fig.),  207,  210,  218 
Photo-micrograph,  214-215 

color  screen  with,  244 

determination  of  magnification    in, 
208,  228 

experiments  at   20, 

to  2000  diameters.,  225-230 


INDEX 


465 


Photo-micrograph,  plates  for,  244 

with  and  without  an  ocular,  230- 

231 

Photo-micrographic    camera,     217-220 
Photo-micrography,  214,  245 

apparatus,  217 

camera  for,  217-219 

compared  with  ordinary  photog- 
raphy, 216 

condenser  for,  228-231 

cover-glass  correction  in,  229 

distinguished  from  micro-photog- 
raphy, 215 

experiments  with,  225-231 

exposure  for,  240 

focusing,  224 

focusing  screen,  210-211 

lighting  for,  222-228,  240 

mechanical  stage  in,  220 

microscope  for,  220 

micrometer  for  magnification  in, 
208,  228 

objectives  and  oculars  for,  220-221 

three  ways  of  bringing  out  details, 
224 

vertical   camera   for,    217-218 

with  and  without  oculars,  226 
Physiologic  histology,  340 
Picric-alcohol,  363 
Picro-fuchsin,  364 
Pillar  of  the  microscope,  27 

pin-hole  card  and  fig.,  9 
Pipette,  334 

for  eggs  and  specimens,  334 
Plane  mirror,  use  of,  54 

plates,  dry,  isochromatic,  pan- 
chromatic, spectrum,  235-237, 
244 

Pleochroism,  268 
Pleurosigma  angulatum,  65,  78 
Point,  burning,  284 

eye,  15,  33 

Pointer  or  indicator  ocular,  102,  195 
Polariscope  and  lighting  for,  264-269 

objects  to  use  for,  266 

objectives  for,  266 

Polarized  light,  ordinary  and  extraor- 
dinary ray,  264 


Polarizer  and  analyzer,  264 
Polarizing  microscope,  264-269 

pedesis    or    Brownian    movement 

with,  119,  268 
Position    of    objects    or    parts,    109- 

no 

Positive  oculars,  23 
Power,  of  microscope,  131 

of  objective,  19,  305 

of  ocular,  308 
Preparation,  of  reagents,  346-366 

of  tissues  and  organs,  368 
Preparations,  cataloguing  and  labeling, 

338-339 

cabinet  for,  340-345 

marked  or  masked  for  projection, 
199-200 

permanent  and  temporary,  321,  332 
Principal  focus,  n,  284 

optic  axis,  n,  282,  284 
Prints,  photographic  for  drawings,  etc., 

178,    181,    234 
Prism,  Amici,  247 

camera  lucida,  162-166 

comparison  in  spectroscope,  249 

for  drawing  with  projection,  185 

Nicol  of  polariscope,  265 
Projection  microscope,   2,  9,   184-189. 
197-203 

history  of,  438-439 

objectives  for,  201 

vertical  microscope  with,  203 

wiring  for  and  fig.,  202 
Pupil  of  lens,  eye-point,  34 
Pupillary  distance  in  binoculars,  91 


Ramsden's  circle,  eye-point,  34 

ocular,  24-25 
Ray  filter  or  color  screen,  237 

compensating  and  contrast,  238 
Razor  for  sections  and  support,  380 
Reagent  board  for  bottles,  345 
Reagents  for  microscopy,  346-367 

preparation  of,  346-367 
Real  image,  13,  34,  133-139,  i?6 
Radiation,  visible  and  invisible  (fig.), 
246,  247,  271 


466 


INDEX 


Record  tables,  232,  337-339.  386,  391 

collodion  method,  391 

negatives,  232 

paraffin  method,  386 
Red,  Congo,  354 

neutral,  362 
Reflected  light,  271 
Reflection,  irregular  and  regular,  272 

total  internal,  275 
Refraction  (fig.),  n,  272,  273-280 

air  to  glass  and  water,  273-274 

index  of,  272 

law  of  sines  in,  272 

medium  in  front  of  objective,  42 

relative  and  absolute  index,  274 
Refractive,  doubly,  263 

highly,  116 

singly,  267 

Relative   position   in   microscopic   ob- 
jects, 109 

index  of  refraction,  274 

of  object  and  image,  14 
Resinous  media,  mounting  in,  328 
Resolution  (eye  and  microscope),  127, 

129,  297 

Resolving  power,  297 
Retinal  image,  5,  7,  9,  130 
Revolving  nose-piece  (fig.),  28 
Ribbon  sections,  379-386 

deparaffming,  384 

drying,  383 

electrification,  381 

spreading,  382 

storing,  381 

tray  for,  342-344 

winder,  382 
Room,  dark  and  drawing   (fig.),   217, 

242 
Royal  Microscopical  Society  standards, 

103-106 
Rule  of  thumb  method,  i,  3 


Sagittal  sections,  409 
Salt  solution,  normal,  363 
Scale,  of  drawing,  309 

size  of  photographs,  208 
wave  length,  248,  250,  255 


Scalpels  (fig.),  379 
Screen,  color,  234-240 

focusing  for  photography,  210-211 
Screw  for  objectives,  R.  M.  S.,  103 
Sealing  cover-glass,  325-327 
Secondary  axis  (optic),  n 
Section  knife  and  sharpening,  372 

lifter  (fig.),  389 

optical,  117 

Sections,  arrangement  on  the  slide,  400, 
404 

clearing,  330 

collodionizing,  387-392 

cutting,  374-381 

dehydration,  229 

deparaffining,  384 

drying,  383 

extending  with  water,  382 

fastening  to  slide,  405 

free-hand,  374 

freezing,  374 

frontal,  407 

longisections,  399 

mounting,  405 

paraffin,  375-386 

ribbon,  379-383 

sagittal,  409 

serial,  399-410 

spreading  or  stretching,  382-383 

spreading  plates  for  (fig.),  382-384 

staining,  392-395,  402 

surface  sections,  400 

thickness  of,  404 

transections,  399 

transferring  (collodion),  390 

vertical,  400 
Serial  sections  and  fig.,  399-410 

arrangement  on  the  slide,  400-404 

cross  or  transections,  406-407 

embryos,  402 

fastening  to  the  slide,  405 

frontal,  407 

imbedding  for,  406-410 

labeling,  410 

mounting  on  slides,  405 

models  from,  410-423 

numbering  slides  of,  400-401 

order  of  sections,  400-404 


INDEX 


467 


Serial  sections,  orienting  objects   for, 

403 

removal  of  mercury  from,  401 

size  of  slides  for,  405 

spreading  on  water,  382 

staining,  392-395 

thickness  of  404 

transections  or  cross  sections,  406- 
407 

transferring  collodion  sections,  370 
S^prpening  section  knives,  372-373 
Shell  vials,  331 
Shellac  cement,  364 
Silver  staining  of  tissues,  365 
Simple  microscope,  7 
Sines,  natural,  Insert  and,  272-279 
Single-objective  binocular,  83-88 
Slides  or  slips  for  specimens  (fig.),  312- 
317 

cleaning,  314-3!  7 

thickness  for  dark-ground  illumina- 
tion, 314 
Slide-basket,  385 
Slide-tray,  199,  342-344 
Slit  mechanism  of  micro7spectroscope, 

254 

Society  screw  for  objectives,  103 
Sodium  lines  and  spectrum,  252-255 
Solar  microscope,  198 
Solar  spectrum,  250,  252 
Soluble  cotton,  353 
Solutions,  346-356 

Farrant's,  356 

how  to  prepare,  346-347 

percentages,  how  to  get,  346 

saturated,  346 

Spectacles,   concave   and   convex,   dis- 
secting, 10,  87,  427-428, 

cylindrical   for  astigmatism,   427- 
428 

history  of,  427 
Spectroscope,  247 

direct  vision,  249 

examination  of  color  screens  with, 

239 
Spectrum,  250-251 

absorption,  250-264 
Angstrom  and  Stokes  law,  252 


banded,  250 

blood,  arterial  and  venous,  259 

carbon  monoxide  hemoglobin,  261 

carmine  solution,  262 

color  screens,  239 

colored  bodies  without  bands,    262 

colorless  bodies,  262 

comparison  or  double,  255 

complementary,  253 

continuous,  248 

daylight  or  sunlight,  250 

double  or  comparison,  255 

hemoglobin,  260 

incandescence  or  line,  250 

methemoglobin,  252 

minerals,  monazite,  etc.,  263 

normal  and  prismatic   (fig.),   247, 
250 

oxy hemoglobin,  260-261 

permanganate  of  potash,  252,  259 

prismatic  and  normal   (fig.),   247, 
250 

sodium,  252 

solar  or  daylight,  252 
Spherical  aberration,  284-285 
Stage,  mechanical,  27,  102 
Stain,  alcoholic  and  aqueous,  393 

acid  and  basic,  393-394 

aqueous  and  alcoholic,  393 

combined,  397 

counter,  392,  395-396 

differential,  392 

elastic,  355 

general,  392 

multiple,  397 

nuclear,  392 
Staining,  332,  392-402 

in  toto,  351 
Stand,  microscopic,  27,  97-100 

focusing  for  embryos,  210 
Standard    distance    for    magnification, 

130,  i39 
Standards,  R.  M.  S.,  for  objectives,  103- 

106 

condensers,  106 
oculars,  106 

Starch,  determination  of,  by  polarized 
light,  268 


468 


INDEX 


Stoke's   and   Angstrom's   law   for   ab- 
sorption spectra,  252-253 
Storing  negatives,  214 

preparations,  340-345 

ribbon  sections,  381 
Strops  and  stropping  for  section  knives, 

372 

Student  or  laboratory  locker,  345 
Style  brief,  Wistar  Institute's,  188 
Substage,  27,  188 

condenser,  57 

Sudan  III  for  fat  staining,  365 
Sulphonal  with  polariscope,  269 
Sulphuric  or  sulfuric  acid,  ether,  320, 356 
Support  of  microtome  knife,  380 
Surface  sections,  400 
Swaying  of  image,  66 
Systems  of  objective,  front,  middle,  and 
back,  20-21 


Table,  black  for,  365 

laboratory  or  student,  55,  95-96 

projection  and  drawing,  179,  186 
Tables,  aperture  of  objectives,  296 

collodion  method,  391 

immersion  liquids,  301 

magnification  and  ocular 

micrometer  valuation,  143 

negative  record,  232 

paraffin  method,  386 

refractive  indices,  277 

size  of  field,  30 
Temporary  mounting,  321 
Tester  for  homogeneous  liquids,  299-301 
Testing  a  camera,  212 

a  microscope  and  its  parts,  97 
Thickness  of  cover-glasses  for  non-ad- 
justable objectives,  285-286 

for  serial  sections,  404 
Tissues,  fixing,  etc.,  368 

washing  apparatus  for,  369 
Towels,  lintless,  315 
Tracing  images  for  drawings,  173,    178, 

181 

Transections  or  cross  sections,  406 
Transferring  collodion  sections,  390 
Transmitted  light,  46 


Tray  for  ribbons  and  slides,  199,  342- 

344 

Tripod  magnifier,  16,  211 
Tube  of  microscope,  27 
Tube-length    (fig.),    27,    79,    286-287 

cover-glass  adjustment  by,  287 
Turn-table  (fig.),  324 

U 

Ultramicroscopy,  75 

Unadjustable  objectives  and  covers  for, 

285-286 

Under-correction,  285,  288 
Unit  of  measure  in  microscopy  (/*),  150 
wave  length  of  light,  256 


Valuation  of  ocular  micrometer,  144-148 
Varnish  for  writing  on  glass,  401 
Varying    magnification    in    compound 

microscope,  139,  157 
ocular  micrometer  valuation,  144, 

157 
Velocity  of  light  and  sine  law,  277 

under  the  microscope,  117 
Velox  printing  paper,  178 
Vertical  camera  (fig.),  210 

microscope,  projection  with,  203 

sections,  400 
Vials,  straight  and  bent-neck,  331 

block  with  holes  for,  332 
Vibrations,  avoidance  in  photography, 

217-218 
Virtual  focus,  12 

image,  8,  9,  15,  133 

standard  distance  at  which  meas- 
ured, 139 

Visible  and  invisible  radiation,  246-247 
Visibility,  127,  234 
Vision,  double,  134 

standard  for  adults,  130 
Visual  angle  (fig.),  6,  9,  127-129 

focus,  222 

necessary  for  resolution,  r  2 7-1 29 

W 

Washing  apparatus  for  tissues,  369 
Watch  glasses,  249,  259 


INDEX 


469 


Water    immersion    objectives.    21,    80, 

296 
Wave    lengths,    designation,    255-256 

scale  of,  255 
Wax  models,  412-413 

paraffin,  363,  375-386 
Wenham's  binocular  microscope  84 
Wire  gauze  experiment  with,  122 
Wiring  for  arc  lamp,  202 
Wistar  Institute  Style  Brief  and  jour- 
nals, 188-190 
Wollaston's  camera  lucida,  162 


Work-room  for  drawing  and  photo-mi- 
crography, 217 
Working  distance  (fig.),  40-43 

free,  39-40 
Writing  diamond,  401 


Xylene,  350 

balsam,  350 
Xylol,  xylene,  350 

Zenker's  fluid,  366 


ADDITIONS 

Angstrom  unit  (A.U.),  150,  256 
Bright-field,  bright-ground,  microscopy, 
§  1250  73 

Centering  and  focusing  the  dark-field 

condenser,§  129  76d 
Chalet  microscope  lamp  (figs.  37-38  506), 

51,  55,  96,  205,  §  129,  76c 
Chylomicrons  (fig.  5of),  §  13  2a  76h 
Condenser,     centering     and     focusing, 

§  129  76d 

Condenser,  dark- field,  68 
Condenser,   parabolic,   paraboloid    (fig. 

48),  72 
Condenser,  reflecting,  §  125,  71,  72 

Dark-field,  dark-ground,  condenser  (fig. 

47-48)  §  122,  125,  69,  72 
Dark-field,    dark-ground    illumination, 

§  117-132,  67-/6g 
Dark-field,     dark-ground     microscopy, 


Dark-field  microscopy,  application,  §131 
Dark-field  microscopy,  steps  in,  §  132 

Edmunds,  J.,§  i32a,  76h 

Gulliver,  G.,  §  1323,  761 

Headlight  lamp  (fig.  50,  c-d)  ,§128  76a-b 

Lamps  for  dark-field  work,  §  128,  75~76c 
Lamp-houses  (figs.  37-38,   50,  c-d-e), 

51,  55,  §  128  76-76c 
Lighting     for     dark-field     microscopy, 

§128,  75~76e 


Micron  Ou),  150,  256 
Millimicron  (mju)  150,  256 
Mistakeless,    electric  connections    (fig. 

50  c-d),  76  a-b 
Molecular  base  of  chyle,  chylomicrons. 

§  1323,  76i 

Moore,  laboratory  desk  (fig.  58),  96 
Moore,  Haemospast,  §  1323,  76h 

Paraboloid,   parabolic,    condenser    (fig. 

48),  72 
"Pointolite,"§  128,  76 

Reade,  Rev.  J.   B.,  dark-field   micros- 

copy i  §  no,  68 
Reducing    diaphragms,    and    aperture 

of,  §  125  a-b,  72-73 


Slides  and  covers  for  dark-field  work, 

§  127,  74 
Step-down   transformer    (fig.   50  c-d), 

§  128,  76  a-b 
Stereopticon  lamps  (fig.  506),  §  128,  /6c 

Transformer  for  no  to  6  volts  (fig.  50 
c-d),  §  128,  76  a-b 

Ultramicroscopy     and     dark-field     mi- 

croscopy, §  13  2b,  763 
Units    of    measure    for    small    things: 

—  micron  GU),  millimicron  (m/z) 

and  Angstrom  units  (A.U.),  150, 

256 

Wenham's    parabolic    condensers    (fig. 
48),  §  125,  71 


INDEX   OF   PROPER   NAMES 

[SEE   ALSO   BIBLIOGRAPHY   AND  COLLATERAL   READINGS.] 


Abbe,  309-310,  434-435,  437,  440 

Adams,  439 

Airy,  428 

Alhazen,  426,  440 

Ailing,  340 

Amici,  437 

Archer  and  Diamond,  216 

Atkinson,  213 

Bacon,  Francis,  439 

Bacon,  Roger,  426-428 

Baker,  439-440 

Barbaro,  427,  438 

Barnard,  84 

Bausch,  E.,  103 

Bausch    &    Lomb    Optical    Company, 

19,  123,  287,  372,  440 
Beale,  2,  123,  124,  126 
Beck,  86-87,  126 
Bernard,  Claude,  367 
Black,  247 
Bleile,  259 
Bock,  427 
Bool,  344 
Borellus,  441 
Born,  413 
Bousfield,  216 
Boyer,  336 

Brewster,  57,  60,  292,  435~437>  44i 
Brown,  A.  J.,  53 
Brown,  R.,  126 
Brown  &  Sharpe,  318 
Brookover,  423 
Burch,  439 
Burr,  423 

Caldwell,  320 

Carpenter-Dallinger,   7,    58,    108,  297, 
427 


Cesi,  7 

Chamot,  67,  71,  75-76,  116,  124,  157- 

158,  264,  277,  313 
Cherubin  d'Orleans,  83,  432 
Comstock,  331 
Cox,  3,  437 

Cramer  Dry  Plate  Co.,  245 
Cunningham,  337 

Daniell,  253 
Descartes,  272,  434 
Dippel,  297,  439 
Dixon,  381 
Dolland,  436 
Donne  et  Foucault,  216 
Dudley  and  Thomas,  367 
Durand,  88 
Dwight,  414 

Eastman  Kodak  Co.,  245 
Edinger,  433 
Emerton,  414 
Ewell,  158-159 

Faber,  7 
Fish,  366 
Freeborn,  352,  359 

Gage,  H.  P.,  53,  205,  244 

Gage,  S.  P.,  339,  343,  365,  4i3,  422 

Galileo,  7 

Gamgee  and  McMunn,  260 

Gordon,  60,  75-76 

Goring  and  Pritchard,  439 

Greenman,  343 

Griffith,  337 

Gundlach  Optical  Co.,  300 

Hardesty  and  Lee,  205 
Harting,  150,  424,  427,  433 


472 


INDEX  OF  PROPER  NAMES 


Hathaway,  423 

Hertzel,  435 

Hitchcock,  364 

Hodge,  340 

Hooke,  127,  129,  434,  439 

Ho  well,  130 

Huygens,  442 

Ives,  F.  E.,  85,  86,  432 
Ives,  H.  E.,  52 

Jansen,  430 
Jevons,  126 
Johnston,  423 

Kingsbury,  204,  376,  383,  403,  413 
Kepler,  430,  431,  438-439 

Leitz,  433 

Lincei,  Academy  of,  7 

Listing,  150 

McClung,  379,  382 
Maddox,  216 
Mall,  413 
Mark,  413 
Mees,  245 
Mercer,  216,  311 
Minot,  340,  371 
Moitessier,  219 
Moore,  96 

Nachet,  432 

Needham  and  Lloyd,  367 
Nelson,  66,  435 
Newcomer,  337 
Newton,  442 
Nichols,  248 

Orndorff,  353 

Pansier,  427 
Pennock,  38,  309,  319 
Pholman,  413 
Piersol,  169 
Pillsbury,  165 
Powell  and  Lealand,  58 
Ptolemasus,  426-427 


Queen  &  Co.,  309 

Ramsden,  24,  34,  434 
Richardson,  158 
Riddell,  83,  432 
Riddle,  365 
Risner,  426 
Robin,  433 
Rogers,  158-159 
Rumsey,  209 
Rutherford,  126 

Schaeffner,  423 

Scheiner,  431,  439 

Schott,  437 

Seneca,  425 

Selligue,  436 

Shadboldt,  216 

Shedd, 311 

Smith,  H.  L.,  299-300,  337 

Smith,  G.  M.,  371 

Snell,  272 

Spencer,  C.  A.,  292,  436 

Spencer  Lens  Co.,  19,  89,  287,  372,  433 

Spitta,  292-293,  297 

Starr,  215 

Sternberg,  216 

Tolles,  83,  437-438 

Walgensten,  438 

Walton,  440 

Ward,  340 

Watson,  58,  278 

Wedgewood  and  Da\y,  216 

Wenham,  78,  83-84,  432 

White,  A.  C.,  424 

Wilson,  215 

Winslow,  35 

Wollaston,  439 

Woodward,  215 

Wright,  Sir  A.  E.,  3,  34,  61,  81,  92,  122, 

159,  301-304,  393 
Wright,  Lewis.  291 

Young,  428 

Zahn,  438 

Zeiss,  19,  59,  432,  437 


Interpolation  with  Natural  Sines:  —  If  one  cannot  find  a  sine  exactly  corresponding  with  an 
angle  in  the  table,  or  an  angle  corresponding  with  a  sine  found  in  solving  a  problem,  the  sine  or 
angle  can  be  closely  approximated  by  the  method  of  Interpolation:  Find  the  sine  in  the  table  nearest 
the  sine  whose  angle  is  to  be  determined.  Get  the  difference  of  the  sines  of  the  angles  greater  and 
less  than  the  sine  whose  angle  is  to  be  determined.  That  will  give  the  increase  of  sine  for  that 
region  of  the  arc  for  15  minutes.  Divide  this  increase  by  15  and  it  will  give  with  approximate  accu- 
racy the  increase  for  1  minute.  Now  get  the  difference  between  the  sine  whose  angle* is  to  be 
determined  and  the  sine  just  below  it  in  value.  Divide  this  difference  by  the  amount  found  neces- 
sary for  an  increase  in  angle  of  1  minute  and  the  quotient  will  give  the  number  of  minutes  the 
sine  is  greater  than  the  next  lower  sine  whose  angle  is  known.  Add  this  number  of  minutes  to  the 
angle  of  the  next  lower  sine  and  the  sum  will  represent  the  desired  angle.  Or  if  the  sine  whose 
angle  is  to  be  found  is  nearer  in  size  to  the  sine  just  greater,  proceed  exactly  as  before,  getting  the 
difference  in  the  sines,  but  subtract  the  number  of  minutes  of  difference  and  the  result  will  give  the 
angle  sought.  For  example,  take  the  case  in  Section  108  where  the  sine  of  the  angle  of  28°  54'  is 
given  as  0.48327.  If  one  consults  the  table  the  nearest  sines  found  are  0.48099,  the  sine  of  28°  45', 
and  0.48481,  the  sine  of  29°.  Evidently  then  the  angle  sought  must  lie  between  28°  45',  and  29°. 
If  the  difference  between  0.48481  and  0.48099  is  obtained,  0.48481  -  0.48099  =  0.00382,  and  if  this 
increase  for  15'  be  divided  by  15  it  will  give  the  increase  for  1  minute;  0.00382  -=-  15  =  0.000254. 
Now  the  difference  between  the  sine  whose  angle  is  to  be  found  and  the  next  lower  sine  is  0.48327 
-0.48099  =  0.00382.  If  this  difference  be  divided  by  the  amount  found  necessary  for  1  minute  it 
will  give  the  total  minutes  above  28°  45',  0.00228  -^  0.000254  =  9.  That  is,  the  angle  sought  is  9 
minutes  greater  than  28°  45'  =  28°  54'. 

Table  of  Metric  and  English  Measures :  — 

Meter  (unit  of  length)  =  100  centimeters;    1000  Kilogram  =  1000  grams;    2.2046  (2  1/5  Ibs.). 

millimeters;    1,000,000  microns  Ox);   39.3700  Yard,  3  feet,  36  inches;  0.9144  meter;  91.4399cm. 

inches;   3.2808  feet.  Foot  =  1/3    yard;     12     inches;    0.3048    meter; 

Centimeter  (cm.)  =  10  millimeters;    10,000    mi-  30.48  cm. 

crons;  0.01  meter;   0.3937  (2/5)  inch.  Inch  =  1/36  yard;  1/12  foot;  2.54  cm.;  25.4mm. 

Millimeter,  (mm.)  =  1,000  microns  GU);   0.1  cm.;  Mile  =  1760  yards;  5280  feet;   1.61  kilometers. 

0.001  meter;    0.03937  (1/25  inch). 

Micron  (unit  of  length  in  micrometry)  (/*)  (§246)  Quart  =  1/4   gallon;    2   pints;    32  fluid  ounces; 

=  0.001,    one    thousandth    of    a    millimeter;  °-947  "'ter  (947  cc.).     (U.  S.  liquid). 

0.000001,  one  millionth  of  a  meter;  0.00003937  Fluid  °unc.e  =  8  nuidrachms;    1/32  of   a  quart; 

(1/25000)  inch.  1/16  pint;    29.574  cubic  centimeters  (30  cc. 

Kilometer  =  lOOo'meters;   0.621  or  5/8  mile.  approximately). 

Ounce    avoirdupois  =  437    1/2    grams;     28.349 

Liter  (unit  of  capacity)  =  1000  cubic  centimeters  grams. 

(or  milliliters) ;    1  quart  approximately.  Ounce  apothecaries  or  Troy  =  480  grains;   31.103 

Gram  (unit  of  weight)  =  1  cc.  of  water;    15.432  grams. 

grains.  Pound  (avoirdupois)  =  16  ounces,  453.6  grams. 

To  Change  from  Centigrade  to  Fahrenheit  and  the  Reverse :  — 

From  centigrade  to  Fahrenheit:  Multiply  the  degrees  centigrade  by  9/5  and  add  32.  Exam- 
ple: 20°  C.  =  20  x  9/5  +  32  or  68°  F. 

From  Fahrenheit  to  centigrade:  Subtract  32  and  multiply  by  5/9.  Example:  77°  F.  =  77  —  32 
X  5/9  or  25°  C. 

To  change  from  centigrade  to  absolute  temperature  and  the  reverse:  Add  273  to  the  degrees  in 
centigrade  and  the  sum  will  be  the  absolute  temperature.  Example.  Ice  melts  at  0°  C.  or  0°  + 
273°  ==  273°  absolute,  and  water  boils  at  100°  C.  or  100°  +  273°  =  373°  absolute.  If  the  abso- 
lute temperature  is  given  subtract  273  and  the  result  will  be  the  temperature  on  the  centigrade 
scale.  Example:  Ice  melts  at  273°  absolute,  273°  -  273°  =  0°,  that  is,  ice  melts  at  0°  C.  See  Fig. 
36,  where  absolute  temperature  is  given. 

American  Manufacturers  and  Dealers  in  Microscopes  and  Microscopical  Supplies: — 

The  Bausch  &  Lomb  Optical  Company,  Roch-  Arthur  H.  Thomas  Company,  Philadelphia  Pa. 

ester,  New  York.  Ernst  Leitz,  Optician,  New  York  City,  N.  Y. 

The  Spencer  Lens  Company,  Buffalo,  New  York.  Eimer  &  Amend,  New  York  City,  N.  Y. 

The   Gundlach   Manhattan   Optical  Company,  The  G.  Cramer  Dry  Plate  Company,  St.  Louis, 

Rochester,  New  York.  Mo.     (Photographic  plates  and  color  screens). 

Joseph  Zentmayer,  Philadelphia,  Pa.  The   Eastman   Kodak    Co.,    Rochester,    N.  Y. 
'Edward  Pennock,  3609  Woodland  Ave.,  Phila:  (Dry  plates,  color  screens,   velox  developing 

delphia,  Pa.  paper). 

The  Corning  Glass  Works,  Corning,  New  York.  Ansco    Company,    Binghamton;     N.  Y.    (Cyco 

(Daylight  glass  and  Pyrex  glass).  developing  papers,  etc.). 

C.  H.  Stocking  Company,  Chicago,  111.  Lumiere  Jougla  Co.,  New  York.    (Autochrome 
Williams  Brown  &  Earle,  Philadelphia,  Pa.  plates). 

When  ready  to  buy  a  microscope  or  supplies  get  the  latest  catalogues  of  the  manu- 
facturers;   then  the  newest  models  can  be  seen  and  the  current  prices  determined. 


TABLE    OF    NATURAL    SINES 

Compiled  from  Prof.  G.  W.  Jones'  Logarithmic  Tables 


MINUTES 


DEGREES  AND  QUARTER  DEGREES  UP  TO  90° 


1°  0.01745 
1°,15'0.02181 

1,30  0.02618 

1,45  0.03054 

2  0.03490 
2,15  0.03926 
2,30  0.04362 
2,45  0.04798 

3  0.05234 
3,15  0.05669 
3,30  0.06105 
3,45  0.06540 

4  0.06976 
4,15  0.07411 
4,30  0.07846 
4,45  0.08281 


6,15  0.10887 


0.12187 


7,45  0.13485 
8        0.13917 


16°,     0.27564'31°,,    0.5150446°,     0.7193461 


0.87462 


160,15'0.27983310,15'0.51877460,15'0.72236|610,15'0.87673 


16,30  0.28402 [31,30  0.5225046,30  0.72537 


16,45  0.2882031,45  0.52621 


17   0.29237 


17,15  0.2965432,15  0.53361 


17,30  0.30071 


77,30  0.97630 

77,45  0.97723 

78  0.97815 

18,15  0.3131633,15  0.5482948,15  0.7460663,15  0.8929878,15  0.97905 

18,30  0.3173033,30  0.5519448,30  0.7489663,30  0.8949378,30  0.97992 


17,45  0.3048632,45  0.5409747,45  0.7402262,45   0.88902 
18        0.3090233        0.5446448        0.7431463        0.89101 


18,45  0.3214433,45  0.55557 


19        0.32557 


19,15  0.3296934,15  0.5628049,15  0.7575664,15   0.9007079,15  0.98245 


19,30  0.33381 


19,45  0.3379234,45  0.5700049,45  0.7632364,45  0.9044679,45  0.98404 


5,30  0.0958520,30  0.35021 


0.1045321        0.35837 


21,15  0.36244 


22        0.37461 


22,45  0.38671 


46,45  0.7283761,45  0.8808976,45  0.97338 


32        0.5299247        0.7313562 


47,15  0.7343262,15   0.8849977,15  0.97534 


32,30  0.5373047,30  0.7372862,30  0.88701 


61,30  0.87882 


0.88295 


34        0.5591949        0.75471 


48,45  0.7518463,45  0.89687 


34,30  0.56641 


0.0871620        0.3420235        0.5735850        0.7660465        0.90631 
5,15  0.0915020,15  0.3461235,15  0.5771550,15  0.7688465,15  0.90814 


49,30  0.76041 


64        0.89879  79        0.98163 


35,30  0.5807050,30  0.7716265,30  0.9099680,30  0.98629 

!/ 


5,45  0.1001920,45  0.3542935,45  0.5842550,45  0.7743965,45  0.9117680,45  0.98700 


36        0.5877951        0.777is|66        0.91355 


36,15  0.59131 


6,30  0.1132021,30  0.3665036,30  0.59482 

6.45  0.1175421,45  0.3705636,45  0.5983251,45  0.78532 


51,15  0.7798866,15  0.91531 


51,30  0.7826l|66,30  0.9170681,30  0.98902 


37        0.60182  52        0.78801 


7,15  0.1262022,15  0.3786537,15  0.6052952,15  0.7906967,15  0.92220 


7,30  0.1305322,30  0.3826837,30  0.6087652,30  0.79335 


37,45  0.61222;52,45  0.79600  67,45  0.92554 


67,30  0.92388 


23        0.3907338        0.6156653        0.7986468        0.92718 


8,15  0.1434923,15  0.39474|38,15  0.6190953,15  0.8012568,15  0.92881 


8,30  0.14781 


64,30  0.9025979,30  0.98325 


66,45  0.9187981,45  0.98965 
67   0.92050  82   0.99027 


1U00029 

2  0.00058 

3  0.00087 
40.00116 
5  0.00145 
60.00175 

7  0.00204 

8  0.00233 

9  0.00262 
100.00291 
110.00320 

12  0.00349 

13  0.00378 

14  0.00407 

15  0.00436 

16  0.00465 

17  0.00495 

18  0.00524 

19  0.00553 

20  0.00582 

21  0.00611 

22  0.00640 

23  0.00669 

24  0.00698 

25  0.00727 

26  0.00756 

27  0.00785 

28  0.00814 

29  0.00844 

30  0.00873 

31  0.00902 

32  0.00931 

33  0.00960 

34  0.00989 

35  0.01018 

36  0.01047 

37  0.01076 
380.01105 
390.01134 
400.01164 

41  0.01193 

42  0.01222 

43  0.01251 
440.01280 

45  0.01309 

46  0.01338 

47  0.01367 

48  0.01396 

49  0.01425 

50  0.01454 

51  0.01483 

52  0.01513 

53  0.01542 
540.01571 

55  0.01600 

56  0.01629 

57  0.01658 

58  0.01687 

590.01716J15,30  0.26724!30,30  0.5075445,30  0.7132560,30  0.8703675,30  0.96815J 
600.0174515,45  0.2714430,45   0.5112945,45  0.71630(60,45  0.8725075,45   0.96923 


76°,  0.97030 
76°,15'0.97134 
76,30  0.97237 


77        0.97437 


78,45  0.98079 


80        0.98481 
80,15  0.98556 


81        0.98769 
81,15  0.98836 


82,15  0.99087 

82,30  0.99144 

82,45  0.99200 

83  0.99255 

83,15  0.99307 


23,30  0.3987538,30  0.6225153,30  0.8038668,30  0.9304283,30  0.99357 


8,45  0.1521223,45  0.4027538,45  0.6259253,45  0.80644 

9        0.15643,24  0.4067439  0.6293254  0.80902 

9,15  0.1607424,15  0.4107239,15  0.6327154,15  0.81157 

9,30  0.1650524,30  0.4146939,30  0.6360854,30  0.81412 

9,45  0.1693524,45  0.4186639,45  0.6394454,45  0.81664 


10 


0.1736525        0.4226240        0.64279,55        0.81915 


10,15  0.17794;25,15  0.4265740,15  0.6461255,15  0.82165 


10,30  0.18224!25,30  0.43051 


40,30  0.64945,55,30  0.82413 


10,45  0.18652!25,45  0.4344540,45  0.65276'55,45  0.8265970,45  0.94409,85,45  0.99725 


11        0.19081126        0.4383741        0.6560656        0.82904 
11,15  0.1950926,15  0.4422941,15  0.65935'56,15  0,83147 


11,30  0.19937:26,30  0.4462041,30  0.6626256,30  0.8338971,30  0.9483286,30  0.99813 
11,45  0.20364|26,45  0.4501041,45  0.6658856,45  0.8362971,45  0.94970,86,45  0.99839 


12        0.2079127        0.4539942        0.6691357        0.83867 
12,15  0.21218127,15  0.4578742,15  0.6723757,15  0.84104 


12,30  0.21644127,30  0.4617542,30  0.6755957,30  0.8433972,30  0.9537287,30  0.99905 

A  f\    A  r      f\  r\r\r\*i/\\  r\*    A  r      r\   A  s  r  s  4     *  >*\    A  r      r\^>rnor\r'7>ic'      ^o^r^ri^ri/ir      rvnr  rr\^  OT    A  r      r\  r*r*o^  -7 


12,45  0.2207027,45  0.46561 
13  0.22495  28  0.46947 
13,15  0.22920;28,15  0.47332 


13,45  0.23769j28,45   0.4809943,45  0.69151 


14        0.24192129 


0.48481 


14,30  0.25038!29,30  0.4924244,30  0.70091 
14,45  0.25460i29,45  0.4962244,45  0.70401 
15  0.2588230  0.5000045  0.70711 


43   0.68200  58   0.84805 


43,15  0.68518 


58,15  0.8503573,15  0.95757,88,15  0.99953 


13,30  0.23345:28,30  0.4771643,30  0.6883558,30  0.8526473,30  0.9588288,30  0.99966 


44        0.6X6659        0.85717 


14,15  0.24615:29,15  0.4886244,15  0.6977959,15  0.85941 


15,15  0.2630330,15  0.5037745,15  0.7101960,15  0.8682075,15  0.96705 


58,45  0.85491 


59,30  0.86163 
59,45  0.86384 
60  0.86603 


68,45  0.93201  !83 ,45  0.99406 


69 


0.93358  84   0.99452 


69,15  0.9351484,15  0.99497 

69,30  0.93667,84,30  0.99540 

69,45  0.93819,84,45  0.99580 

70  0.93969,85  0.99619 

70,15  0.9411885,15  0.99657 

70,30  0.9426485,30  0.99692 


71   0.9455286   0.99756 
71,15  0.9469386,15  0.99786 


72   0.9510687   0.99863 
72,15  0.95240|87,15  0.99885 


42,45  0.6788057,45  0.8457372,45  0.9550287,45  0.99923 


73   0.95630  88   0.99939 


73,45  0.96005,88,45  0.99976 

74  0.9612689  0.99985 
74,15  0.9624689,15  0.99991 
74,30  0.9636389,30  0.99996 
74,45  0.9647989,45  0.99999 

75  0.96593,90  1.00000 


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